KR102679112B1 - Probe Set for Isothermal One-Pot Reaction using Split T7 promoter and Uses Thereof - Google Patents

Abstract

The present invention relates to a probe set for an isothermal single reaction using a split T7 promoter and its use. The split T7 promoter is newly introduced into a three-way junction construct, and a large amount of fluorescent RNA aptamer is generated in the presence of a target molecule at isothermal. It is designed to produce nucleic acid biomarkers quickly and easily with only one enzyme in one tube. A variety of applications are possible through high-sensitivity detection of multiple nucleic acid biomarkers and detection in which analysis is performed without an additional nucleic acid extraction process. Not only can multiplex analysis be performed in one tube, but it can also detect various molecules, including viruses and pathogens. It is excellent because it allows for diagnosis.

Description

Probe Set for Isothermal One-Pot Reaction using Split T7 promoter and Uses Thereof}

This technology is about a system that quickly and conveniently detects various target molecules. It is designed to generate a large amount of fluorescent RNA aptamers in the presence of target molecules at isotherm by introducing a new split T7 promoter into a 3-way splicing construct. Accordingly, the limitations of existing on-site diagnosis can be overcome, and various applications are possible through high-sensitivity detection of multiple target molecules and detection in which analysis is performed without an additional extraction process.

In December 2019, a new respiratory infectious RNA virus, severe acute respiratory syndrome coronavirus (SARS-CoV-2), and its mutant viruses emerged and rapidly spread around the world. Therefore, there is a need for a technology to quickly and conveniently detect SARS-CoV-2 to suppress the spread of the virus.

Currently, the most commonly used method for detecting SARS-CoV-2 is qRT-PCR (quantitative reverse transcription polymerase chain reaction) based on nucleic acid amplification. Various primer-probe sets have been identified and have the advantage of having very sensitive detection limits, but all PCR tests require temperature control and are performed in specialized laboratories, so they take a long time (1-2 days) and are expensive. do.

Isothermal nucleic acid amplification methods using various strand displacement polymerases have been developed to compensate for the shortcomings of these temperature control-based methods and to improve testing convenience. The isothermal amplification method was developed to compensate for the shortcomings of temperature control-based methods and to facilitate point-of-care testing. However, most isothermal amplification methods require a denaturation/annealing step through temperature control before starting the reaction. In addition, there is a disadvantage that it is difficult to optimize the reaction to various enzymes and the detection step is complicated. In particular, in the case of SARS-CoV-2, most isothermal amplification methods are carried out by converting RNA into cDNA, and since DNA amplification products are produced in large quantities, the converted cDNA and amplified DNA are not easily degraded and are vulnerable to contamination. do. Such contamination can interfere with the reaction and result in high false positives. Therefore, a fast single-step isothermal amplification method is still needed to detect SARS-CoV-2.

Signal-mediated amplification of RNA technology (SMART) based on the three-way junction structure can be applied to gDNA, total RNA, etc., and has been used for highly sensitive detection of various nucleic acid biomarkers. However, despite the excellent advantages of SMART technology, it has the limitation that it requires two enzymes, DNA polymerase and RNA polymerase, and when the two enzymes coexist, a non-specific signal is generated, making a one-step reaction impossible. The downside is that it takes a long time to react.

Meanwhile, fluorescent RNA aptamers have high specificity and affinity for specific molecules and have the characteristic of producing a high fluorescence signal when bound. Because it has higher thermal and chemical stability compared to protein antibody molecules, it is widely used in not only intracellular experiments but also extracellular experiments. These fluorescent RNA aptamers have the advantage of being inexpensive, have low background signal, and can be applied to multiplex analysis.

Regarding SMART technology, International Patent Publication WO 99/37806 discloses SMART technology for detecting target nucleic acid sequences, but the target nucleic acid was detected using two polymerases, and only one polymerase was used. The technology has not been disclosed.

Accordingly, the present inventors completed the present invention by focusing on target detection using a three-way conjugation structure that is highly specific and can easily distinguish changes in the target molecule while using a single enzyme.

Accordingly, the present inventors worked to develop a new isothermal nucleic acid amplification technology that can quickly detect target molecules while overcoming the shortcomings of existing SMART analysis. As a result, a new isothermal nucleic acid amplification based on a three-way junction structure was developed. technology (37°C) was invented and a probe was designed inexpensively and easily without additional labeling to enable multiple analysis.

Accordingly, an object of the present invention is to provide an isothermal one-pot reaction probe set for detecting one or more target molecules, including a first probe and a second probe.

Another object of the present invention is to provide a composition for detecting a target molecule including an isothermal single reaction probe set for detecting a target molecule including the first probe and the second probe.

Another object of the present invention is to provide a kit for detecting a target molecule, including the composition for detecting the target molecule, a polymerase, and an isothermal single reaction solution.

Another object of the present invention is to provide a method for detecting a target molecule using the isothermal single reaction probe set for detecting the target molecule.

Another object of the present invention is to provide an on-site molecular diagnostic method using an isothermal single reaction probe set for detecting target molecules.

The terms used herein are for descriptive purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that it does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof. Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the technical field to which the embodiments belong. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless explicitly defined in the present application, they should be interpreted in an ideal or excessively formal sense. It doesn't work.

To achieve the above object, the present invention provides an isothermal one-pot reaction probe set for detecting one or more target molecules, comprising the following first and second probes:

The first probe is a promoter probe (PP) having a structure comprising the following general formulas I and II,

[General formula I]

5'-X-Y-Z-3'

[General formula II]

3'-Ya-5'

In the general formula I,

X is a region of the aptamer sequence with an interactive labeling system comprising one label or multiple labels that generates a detectable signal;

Y is a sequence complementary to the T7 promoter;

Z is a site that binds or connects to the target molecule;

X and Y are deoxyribonucleotides;

In the general formula II,

Ya is a partial sequence of the T7 promoter; Ya is a deoxyribonucleotide;

The second probe is a promoter probe (PP) having a structure of the following general formula (III),

[General formula III]

3'-Yb-Z'-5'

In the general formula (III),

Yb is absent, is a partial sequence of the T7 promoter, and is a deoxyribonucleotide;

Z’ is a site that binds or connects to the target molecule;

The first probe and the second probe are characterized in that after binding or linking to the target molecule, transcription is initiated by a polymerase to generate a signal.

In one embodiment of the present invention, the target molecule may be nucleic acid, protein, cell, and ATP.

In one embodiment of the present invention, when the target molecule is a nucleic acid, Z and Z' are nucleic acid sequences that bind complementary to the nucleic acid to be detected, and when the target molecule is a protein, Z and Z' are the It is an antibody or aptamer that can bind to a protein, and when the target molecule is a cell, Z and Z' are any one selected from the group consisting of extracellular protein, cellular phospholipid, bacterial peptidoglycan, and LPS. If it is an antibody or aptamer that can bind to the above, and the target molecule is ATP, Z and Z' may be characterized as split aptamers that can bind to ATP.

In one embodiment of the present invention, Ya and Yb are partial sequences of the T7 promoter, and when Ya and Yb are sequentially arranged in the order of Ya and Yb, they form the T7 promoter, and the Ya:Yb split ratio is It may be characterized as 20:0 to 15:5.

In a preferred embodiment of the present invention, the Ya:Yb split ratio may be 16:4.

In one embodiment of the present invention, the label may be selected from the group consisting of a chemical label, an enzyme label, a radioactive label, a fluorescent label, a luminescent label, a chemiluminescent label, and a metal label.

In one embodiment of the present invention, the general formula I of the first probe may be modified into general formula I', which further includes an overlapping sequence W between Y and Z;

[General formula I']

5'-X-Y-W-Z-3'

The general formula III of the second probe is characterized in that it can be transformed into general formula III', which additionally includes an overlapping sequence W' between Yb and Z'.

[Formula III']

3'-Yb-W'-Z'-5'

It may be an isothermal single reaction probe set for detecting target molecules.

In one embodiment of the present invention, the length of the overlapping sequences W and W' may be 1bp or 2bp.

In one embodiment of the present invention, the isothermal single reaction may be performed at a constant temperature in the range of 15°C to 50°C.

In one embodiment of the present invention, the probe set may be characterized in that it detects circular RNA, distinguishing it from linear RNA.

In one embodiment of the present invention, the polymerase is bacteriophage T7 RNA polymerase, bacteriophage T3 polymerase, bacteriophage RNA polymerase, bacteriophage ΦII polymerase, Salmonella bacteriophage sp6 polymerase, Pseudomonas bacteriophage gh-1 polymerase, Escherichia coli ( E. coli RNA polymerase holoenzyme, E. coli RNA polymerase core enzyme, human RNA polymerase I, human RNA polymerase II, human RNA polymerase III, human mitochondrial RNA polymerase and their It may be selected from the group consisting of variants.

In one embodiment of the present invention, the isothermal single reaction is unified into a single reaction solution containing Tris-HCl, MgCl2, DTT, spermidine, rNTPs, RNase inhibitor, and SSB (Single-Stranded DNA Binding Protein) and carried out simultaneously. It can be characterized as being performed.

The present invention also provides a composition for detecting a target molecule, including the isothermal single reaction probe set for detecting the target molecule.

In one embodiment of the present invention, the composition may include an isothermal single reaction probe set for detecting two or more target molecules.

In one embodiment of the present invention, the isothermal single reaction probe sets for detecting two or more target molecules each include different interactive labeling systems; Each of the two or more probe sets binds to a different target molecule; This may enable multiple detection of different target molecules.

According to another preferred embodiment of the present invention, the isothermal single reaction may be performed at a constant temperature in the range of 15°C to 50°C.

The present invention also provides a kit for detecting a target molecule, including the composition for detecting the target molecule, a polymerase, and an isothermal single reaction solution.

Additionally, the present invention provides a method for detecting a target molecule using the isothermal single reaction probe set for detecting the target molecule.

In one embodiment of the present invention, the method of detecting the target molecule may include performing an isothermal nucleic acid amplification reaction.

According to a preferred embodiment of the present invention, the isothermal nucleic acid amplification reaction may be RPA (Recombinase Polymerase Amplification).

The present invention also provides an on-site molecular diagnostic method using the isothermal single reaction probe set for detecting the target nucleic acid sequence.

In one embodiment of the present invention, the molecular diagnostic method can be used to detect pathogenic microorganisms.

According to another preferred embodiment of the present invention, the pathogenic microorganisms include Staphylococcus Aureus, Vibrio vulnificus, E. coli, and Middle East respiratory syndrome coronavirus. East Respiratory Syndrome Coronavirus, Influenza A virus, Severe Acute Respiratory Syndrome Coronavirus, Respiratory Syndrome Virus (RSV), Human Immunodeficiency Virus (HIV), Herpes Simplex Virus ( HSV), human papillomavirus (HPV), human parainfluenza virus (HPIV), dengue virus, hepatitis B virus (HBV), yellow fever virus, rabies virus, plasmodium, cytomegalovirus (CMV), Mycobacterium tuberculosis, Chlamydia trachomatis, rotavirus, human metapneumovirus (hMPV), Crimean-Congo hemorrhagic fever virus, Ebola virus, Zika virus, Henipa virus, norovirus, Lassa Viruses, rhinovirus, flavivirus, Rift Valley fever virus, hand, foot and mouth disease virus, Salmonella sp., Shigella sp., Enterobacteriaceae sp., Pseudomonas sp., Morag It may be one or more species selected from the group consisting of Moraxella sp., Helicobacter sp., and Stenotrophomonas sp.

The present invention enables rapid and simple detection of target molecules with only one enzyme in one tube. In addition, not only is the design of the probe simple and does not require additional labeling, but it also provides an isothermal nucleic acid amplification method that allows the implementation of a direct system without pretreatment through heat treatment alone. In addition, multiplex analysis is possible in one tube, and various molecular diagnoses, including viruses and pathogens, are possible.

Figure 1 shows a schematic diagram of Split T7 promoter-based isothermal Amplification with RNA Aptamer (hereinafter referred to as “STAR”) for detecting target molecules. The three important components of the STAR reaction of the present invention are the STAR DNA probe, T7 RNA polymerase, and fluorescent dye. In the presence of target RNA, the STAR DNA probe forms a three-way junction structure, resulting in a double-stranded T7 promoter with a nick site. T7 RNA polymerase can bind to the T7 promoter and initiate transcription to generate RNA aptamers that emit large amounts of light. Afterwards, the RNA aptamer combines with a fluorescent dye to generate a highly enhanced fluorescence signal. One-step reaction occurs within 30 minutes at 37°C.
Figure 2 shows a schematic diagram when the target molecule is a protein or cell.
Figure 3 shows the results of splitting of the split T7 promoter at various ratios.
Figure 4 shows the optimization results of the overlap length.
Figure 5 shows the results regarding the stability of the T7 promoter when a bulge structure is added to the 3-way junction structure.
Figure 6 shows the concentrations of DTT and MgCl2 in the composition of the buffer for optimization of reaction conditions.
Figure 7 shows the fluorescence intensity results measured to find the optimal concentration of spermidine and single-stranded DNA binding protein (SSB).
Figure 8 shows fluorescence intensity results according to T7 RNA polymerase concentration, reaction time, reaction temperature, and cooling method.
Figure 9 shows the results of comparing the transcription efficiency of the T7 promoter with a 3-way spliced structure and a linear structure when using an optimized buffer.
Figure 10 shows gel electrophoresis results to confirm the three-way junction structure.
Figure 11 is a fluorescence spectrum result without each element of STAR.
Figure 12 is a schematic diagram showing that it can be used as a method to detect circular RNA, distinguishing it from linear RNA.
Figure 13 shows results showing that the detection intensity increases when an isothermal nucleic acid amplification reaction is performed before performing the STAR reaction.
Figure 14 relates to the detection sensitivity of each locus of the SARS-CoV-2 N gene, Figure 14A shows a schematic diagram of a portion of the SARS-CoV-2 N gene and the locus (1-5) of the target RNA, and Figure 14B is a heatmap showing the detection sensitivity of STAR using five sets of DNA probes for different loci (L1-L5) of the N gene, Figure 14C is the detection limit of STAR at L2 and L4, with the STAR probe set at each locus. It is 100nM.
Figure 15 shows a combination of two sets of STAR probes to improve sensitivity. Figure 15A is a schematic diagram of the SARS-CoV-2 N gene and two other loci of other human coronaviruses, with nucleotides of SARS-CoV-2 and other viral RNA sequences shaded in gray. Figure 15B is an agarose gel electrophoresis image of the STAR probe set to N gene RNA, where the concentrations of the L2 and L4 probe sets are 500 nM each (a and b), and when the L2 and L4 probes are present simultaneously, the concentration of each probe set is It is 250nM(c). Figure 15C shows fluorescence signals measured in the presence of the L2 probe set (100 nM), the L4 probe set (100 nM), and both the L2 and L4 probe sets (50 nM+50 nM), with a concentration of target N gene RNA of 1 nM. Figure 15D is the detection limit of using the STAR probe set combination at L2 and L4, where the concentration of the STAR probe set is 50 nM at each other locus. Figure 15E shows the detection specificity of the STAR assay when using two STAR probe sets (L2 and L4), where the concentration of SARS-CoV-2 N gene RNA was 1 nM and the others were 100 nM. The concentrations were each 50nM.
Figure 16 shows the application of STAR for detecting SARS-CoV-2 D614G mutation. Figure 16A shows a schematic diagram of STAR analysis for the D614G mutation in the S gene of SARS-CoV-2, with the mutation site in yellow and the mismatch sequence shown in red to avoid false positive signals from the wild-type target. Figure 16B shows malachite green (MG) signal intensity obtained after STAR using different nucleobases (A, T, G and C) at the N4 position. Figure 16C is the detection limit of STAR for the D614G mutation, with concentrations of target RNA ranging from 1 nM to 100 fM. Figure 16D shows the detection results of the D614G mutation in mixtures of mutant/wild targets with different molar ratios (0%, 0.1%, 1%, 10%, 50% and 100%).
Figure 17 shows a system for multiple detection. Figure 17A is a schematic diagram of a one-pot multiplex STAR for simultaneous detection of SARS-CoV-2 N gene and D614G mutation. Figure 17B is the normalized fluorescence emission spectra of TO1-biotin and MG binding to mango aptamer and MG aptamer, respectively. Figure 17C is a heatmap showing the results of one-pot, multiplex detection of the N gene and D614G mutation, and the concentration of target RNA is 10nM. Figure 17D shows the results of one-pot multiplex detection of N gene and D614G mutation at different concentrations.
Figure 18 shows a system for pathogen detection. Figure 18A is a schematic diagram of pathogen detection using STAR. For pathogen detection, STAR probes were designed for the 16s rRNA region of the pathogen, (i) one was STAR using extracted total RNA and (ii) the other was pyrolysis. It is a direct STAR using cell lysate obtained through Figure 18B: Detection of bacterial 16S rRNA using STAR. Total RNA concentrations ranging from 100 pg/μL to 100 fg/μL were tested. Figure 18C is a heatmap showing the results of direct STAR for detection of different pathogens, where the concentration of each pathogen is 10 ³ cells/μL.

As described above, while using an isothermal amplification reaction, there has been a need to overcome the disadvantages that non-specific signals are generated when two enzymes are used, a one-step reaction is not possible, and the reaction time is long. As a result of our efforts to develop a new isothermal amplification technology that can quickly detect target molecules while overcoming the shortcomings of existing SMART analysis, the present inventors have developed a new isothermal amplification technology based on 3-way junction that can quickly analyze target molecules. invented. In the invention according to the present invention, the transcription reaction by the target substance is designed to occur effectively by controlling the division ratio of the T7 promoter sequence. Based on this, the T7 promoter based on the three-way junction structure is formed only when the target molecule is present. This made it possible to obtain an amplified signal using only one polymerase in one tube. In addition, to further increase sensitivity, an additional three-way bonding structure was designed to enable binding to multiple regions in the target molecule, and through this, an increase in sensitivity was achieved. In the present invention, when the target molecule is a nucleic acid sequence, changes in the sequence can be easily distinguished very specifically, and it has the characteristic of amplifying a sequence dependent on the target molecule rather than amplifying the target molecule as is. Based on this, By designing two different types of light-up RNA aptamers, we implemented multiplex analysis that can simultaneously identify two or more molecules in one tube. In addition, based on the excellence of the present invention, it has been verified that a method can be applied to the detection of various pathogens to successfully detect nucleic acid biomarkers of pathogenic microorganisms without an additional nucleic acid extraction process, so it is simpler, faster, and more sensitive than the existing method. It is excellent because it can be detected easily.

Hereinafter, the present invention will be described in more detail.

The present invention is an isothermal one-pot reaction probe set for detecting one or more target molecules, including a first probe and a second probe,

The first probe is a promoter probe (PP) having a structure comprising the following general formulas I and II,

[General formula I]

5'-X-Y-Z-3'

[General formula II]

3'-Ya-5'

In the general formula I,

X is a region of the aptamer sequence with an interactive labeling system comprising one label or multiple labels that generates a detectable signal;

Y is a sequence complementary to the T7 promoter;

Z is a site that binds or connects to the target molecule;

X and Y are deoxyribonucleotides;

In the general formula II,

Ya is a partial sequence of the T7 promoter; Ya is a deoxyribonucleotide;

The second probe is a promoter probe (PP) having a structure of the following general formula (III),

[General Formula III]

3'-Yb-Z'-5'

In the general formula (III),

Yb is absent, is a partial sequence of the T7 promoter, and is a deoxyribonucleotide;

Z’ is a site that binds or connects to the target molecule;

The first probe and the second probe are hybridized with the target molecule, and then transcription is initiated by a polymerase to generate a signal,

An isothermal single reaction probe set for detecting target molecules is provided.

The first probe of the probe set of the present invention contains one oligonucleotide molecule, each with a structure that includes three different portions within one oligonucleotide molecule: a label that generates a detectable signal; or X is an aptamer sequence region with an interactive labeling system containing multiple labels, Y is a sequence complementary to the T7 promoter, and Z is a region that binds or connects to a target molecule; Ya, a partial sequence of the T7 promoter complementary to Y.

In addition, the second probe of the probe set of the present invention also has a structure containing one or two unique different sites within one oligonucleotide molecule: complementary to Y, a partial sequence of the T7 promoter, and a sequence contiguous with Ya. Yb with; Z', the site that binds or connects to the target molecule. Yb may not exist when it is 20:0 depending on the split ratio described later.

This structure allows the probe set of the present invention to be a probe that exhibits high detection effectiveness in a short time under isothermal single reaction conditions in a single step.

In the present invention, the term "probe" binds to a target nucleic acid of a complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation, and thus forms a duplex structure. It is a nucleic acid that can A probe binds or hybridizes to a “probe binding site”. In particular, the probe may be labeled with a detectable label to facilitate detection of the probe once it has hybridized to its complementary target. However, alternatively, the probe may be unlabeled, but detectable directly or indirectly by specific binding to the labeled ligand. Probes can vary considerably in size. Typically probes are at least 7 to 15 nucleotides in length. Other probes are at least 20, 30 or 40 nucleotides in length. Other probes are somewhat longer, being at least 50, 60, 70, 80, or 90 nucleotides in length. Other probes are even longer, at least 100, 150, 200 or more nucleotides in length. The probe may also be of any length within any range defined by any of the above values (e.g., 15-20 nucleotides in length).

In the present invention, the term "molecule" refers to all types of biomolecules or cells to be detected, and biomolecules are target molecules that constitute living organisms and are responsible for their functions, the most common of which are proteins, nucleic acids, polysaccharides, and lipids. Large macromolecules and low molecular substances include organic substances such as amino acids, nucleotides, monosaccharides, and vitamins, metals such as iron and copper, and inorganic ions. It includes biomolecules extracted from the natural environment, biomolecules produced synthetically, and biomolecules modified by attachment of initiation factors. In the present invention, nucleic acids, proteins and cells are preferred.

In the present invention, the term "target nucleic acid sequence" refers to all types of nucleic acids to be detected, and includes chromosomal base sequences from different species, subspecies, or variants, or chromosomal mutations within the same species. This may include, but is limited to, all types of DNA, including genomic DNA, mitochondrial DNA, and viral DNA, or all types of RNA, including mRNA, ribosomal RNA, non-coding RNA, tRNA, and viral RNA. It doesn't work.

As used herein, the term “oligonucleotide” refers to an oligomer of nucleotides. As used herein, the term “nucleic acid” refers to a polymer of nucleotides. As used herein, the term “sequence” refers to the nucleotide sequence of an oligonucleotide or nucleic acid. The oligonucleotide or nucleic acid may be DNA, RNA, or an analog thereof (eg, a phosphorothioate analog). The oligonucleotide or nucleic acid may also include modified bases and/or backbones (e.g., modified phosphate linkages or modified sugar moieties). Non-limiting examples of synthetic backbones that impart stability and/or other advantages to nucleic acids may include phosphorothioate linkages, peptide nucleic acids, locked nucleic acids, xylose nucleic acids, or analogs thereof.

The term nucleic acid includes, for example, genomic DNA; Complementary DNA (cDNA) (this is a DNA representation of mRNA, usually obtained by reverse transcription or amplification of messenger RNA (mRNA)); A DNA molecule produced synthetically or by amplification; and any form of DNA or RNA, including mRNA.

The term nucleic acid includes single-stranded molecules as well as double- or triple-stranded nucleic acids. In a double or triple stranded nucleic acid, the nucleic acid strands need not be coextensive (i.e., a double stranded nucleic acid need not be double stranded along the entire length of both strands).

The term nucleic acid also includes any chemical modifications thereof, such as by methylation and/or capping. Nucleic acid modifications can include the addition of chemical groups containing additional charge, polarizability, hydrogen bonding, electrostatic interactions, and functionality to individual nucleic acid bases or to the nucleic acid as a whole. These modifications include sugar modifications at the 2' position, pyrimidine modifications at the 5 position, purine modifications at the 8 position, modifications at cytosine exocyclic amines, substitution of 5-bromo-uracil, main chain modifications, and isobases isocytidine and isoguanidine. It may include base modifications such as specific base pair combinations.

More specifically, in the probe design of the present invention, two single-stranded DNA probes are designed so that the target recognition site is exposed, enabling isothermal hybridization of the target molecule and the probe set.

The “site that binds or connects to the target molecule” included in the probe refers to a sequence that is complementary to a portion of the nucleic acid sequence of the target molecule and can hybridize with the target molecule when the target molecule is a nucleic acid. For example, the “site that binds or connects to the target molecule” may have a sequence complementary to a portion of the target molecule by at least 90%, preferably at least 95%. If the target molecule is a protein, the “region that binds or connects to the target molecule” means a region that can bind or connect to the protein, which is the target molecule, in the aptamer acting as a probe. The “site that binds or connects to the target molecule” can have its length appropriately adjusted depending on the target molecule, and is not limited to a specific length. For example, if the target molecule is a miRNA, the "site that binds or connects to the target molecule" may consist of around 22 nucleotides, but if the target molecule is mRNA or protein, the "site that binds or connects to the target molecule" may be composed of around 22 nucleotides. The “part” may be shorter or longer than that.

In the present invention, the term “hybridization” refers to the formation of a double-stranded nucleic acid by hydrogen bonding between single-stranded nucleic acids with complementary base sequences, and is used in a similar sense to annealing. However, in a slightly broader sense, hybridization includes cases where the base sequences between two single strands are completely complementary (perfect match) as well as exceptional cases where some base sequences are not complementary (mismatch).

As used herein, the term “complementary” refers to the ability for correct pairing between two nucleotides. That is, if a nucleotide at a given position of a nucleic acid can form a hydrogen bond with a nucleotide of another nucleic acid, the two nucleic acids are considered complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules may be “partial,” where only a portion of the nucleotides are bonded, or complementarity may be complete, when total complementarity exists between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

When the target molecule is a target nucleic acid sequence, the first probe binds to the Upstream Hybridization Sequence (UHS) of the target nucleic acid sequence, has a sequence complementary to the T7 promoter, and has an aptamer sequence. Ya included in the general formula II of the first probe corresponds to a partial sequence of the T7 promoter, and Yb in the general formula III of the second probe also corresponds to the remaining partial sequence of the T7 promoter, and Ya and Yb are continuous sequences and form one sequence. It constitutes the T7 promoter and corresponds to two divided fragments. In the presence of target RNA, the probe set forms a three-way junction structure, resulting in a double-stranded T7 promoter with a nick site. Therefore, if the target nucleic acid sequence does not exist, the T7 promoter sequence is split into Ya and Yb, so the polymerase cannot bind. Transcription of the tamer site is initiated (see Figure 1).

Since a signal is generated in response to a specific substance by the aptamer sequence region X of the first probe, the final product can be confirmed.

When the target molecule is a protein or cell, the first probe and the second probe each have a site that binds or connects to the target protein or cell. For example, as shown in FIG. 2, an antibody or aptamer that specifically binds to a target site of a protein or cell may exist in a bound or linked form. In the case of aptamers, they can be linked through complementary sequences such as oligonucleotides (DNA, etc.) of a certain length (~several tens of nucleotides), and in the case of antibodies, they can be linked by NHS (N-hydroxysuccinimide) ester synthesis, etc.

As in the case where the target nucleic acid sequence is present, in the presence of the target protein or cell, the probe set forms a three-way junction structure and a double-stranded T7 promoter with a nick site is completed. Therefore, when the target protein is not present, the T7 promoter sequence is split into Ya and Yb, so the polymerase cannot bind, and only when the target protein or cell is present, the polymerase binds to increase the pressure of the first probe. Transcription of the tamer site is initiated (see Figure 2).

In the present invention, the "Aptamer" used as a reporter is a single-stranded nucleic acid (DNA, RNA or modified) that has the characteristic of being able to bind to the target molecule with high affinity and specificity while having a stable tertiary structure itself. nucleic acid).

Since the development of the aptamer discovery technology called SELEX (Systematic Evolution of Ligands by EXponential enrichment), aptamers that can bind to various target molecules, including low-molecular organic substances, peptides, and membrane proteins, have been continuously discovered. Aptamers are often compared to single antibodies due to their inherent high affinity (usually at the pM level) and the ability to bind to target molecules with specificity, and in particular, they have high potential as alternative antibodies to the extent that they are called “chemical antibodies.” In addition, the binding of an aptamer to a specific chemical molecule can be confirmed through methods such as fluorescence, which varies depending on the binding material, and the absorption and emission wavelength bands are different.

Any type of aptamer and reactive material that interacts with the aptamer of the present invention can be used as long as it can generate a detectable signal as the desired effect.

According to a preferred embodiment of the present invention, when the target molecule is a target nucleic acid sequence, when the first probe and the second probe hybridize with the target nucleic acid sequence, the first probe and the second probe are immediately adjacent to each other. ) is located in the location.

Y of the first probe; Ya of the first probe and Yb of the second probe are hybridized, and one T7 promoter structure is formed. The term "adjacent" used herein when referring to the hybridization position of the first probe and the second probe means that the ends of Ya and Yb in the two probes are located so that they can be connected to each other, and Ya and Yb are hybridized with Y. This means that they are close enough together to be possible.

In one embodiment of the present invention, the target molecule is a protein, peptide, nucleic acid, carbohydrate, lipid, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, pathogen, toxic substance, substrate, metabolite, Can be, but is not limited to, ATP, cocaine, mercury, transition state analogs, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells, tissues, etc., and almost any chemical or biological actuator. It will be an effector and refers to a target molecule that can be used for molecules of any size. According to a preferred embodiment of the present invention, the target molecule may be nucleic acid, protein, cell, and ATP.

According to a preferred embodiment of the present invention, when the target molecule is a nucleic acid, Z and Z' are nucleic acid sequences that bind complementary to the nucleic acid to be detected, and when the target molecule is a protein, Z and Z' are the It is an antibody or aptamer capable of binding to a protein, and when the target molecule is a cell, Z and Z' are any one selected from the group consisting of extracellular protein, cellular phospholipid, bacterial peptidoglycan, and LPS. If it is an antibody or aptamer that can bind to the above, and the target molecule is ATP, Z and Z' may be a split aptamer that can bind to ATP.

Specifically, when the target molecule is ATP, preferably, a substance such as ATP corresponds to a small molecule, and Z and Z' may be separate aptamer sites capable of binding to ATP. . In detail, the paper A. Chen et al. Please refer to the content disclosed in "Split aptamers and their applications in sandwich aptasensors" Trends in Analytical Chemistry 80 (2016) 581-593. As described herein, when the target molecule is ATP, it means that the DNA aptamer that binds to intact ATP can be cut in half to create a two-strand DNA aptamer (Split DNA aptamer) and used by a method known in the art. It can be used depending on

Representative sites that bind or connect to target molecules include antibodies, peptides, nucleic acids, and aptamers.

An antibody is a substance that specifically binds to the epitope of an antigen and causes an antigen-antibody reaction. An aptamer refers to a small (20-60 nucleotide) piece of single-stranded nucleic acid (DNA or RNA) that has the property of being able to bind specifically and with high affinity to various types of substances, from small molecular compounds to proteins.

The probe is a linker region bound between an aptamer sequence region having an interactive labeling system including one label or multiple labels that generate the detectable signal and a region that binds or connects to the target molecule. may additionally be included. In the present invention, the linker region may be introduced at one end of the probe to reach a site that binds or connects to a target molecule. The specific type of the linker region is not particularly limited, but for example, the linker region may be a short nucleic acid sequence of 4 to 10 nucleotides.

According to a preferred embodiment of the present invention, in the probe set of the present invention, Ya and Yb are partial sequences of the T7 promoter, and when Ya and Yb are sequentially arranged in the order of Ya and Yb, the T7 promoter and the Ya:Yb split ratio may be 20:0 to 15:5.

Since the T7 promoter sequence is divided into the first and second probes, if the target molecule is not present, the polymerase cannot bind to the T7 promoter and initiate transcription, and ultimately, the separation of the first and second probes Since no signal is generated from the label on the transcription product, detection of the target molecule is not achieved. When a target molecule is present, Ya of the first probe and Yb of the second probe are sequentially arranged to form a T7 promoter, and the transcription process is initiated by polymerase. When the transcription process is initiated, a product of X of the first probe containing the aptamer sequence may be generated.

Existing SMART (Signal mediated amplification of RNA technology) technology used DNA polymerase, and then RNA polymerase had to be used sequentially to use two enzymes. Accordingly, when two enzymes exist together, a non-specific signal is generated. Because this occurred, a one-step reaction was impossible, and the reaction time also took a long time. However, the present invention omitted the step of generating the T7 RNA promoter using DNA polymerase, and instead split the T7 promoter to prevent the T7 promoter itself from being generated when the target does not exist. Through this, a system was introduced that can detect target molecules with only one polymerase. Since only one enzyme is used, the occurrence of non-specific signals is reduced and the reaction time is shortened, enabling rapid reaction.

That is, since the core of the present invention is the effective formation of a split T7 promoter in the presence of a target molecule by inducing a three-way junction structure, in the present invention, the existing T7 promoter was split and the transcription efficiency according to each ratio was analyzed. Specifically, in one embodiment of the present invention, in Example 1-1, in order to control the formation of the T7 promoter, nick sites were sequentially inserted starting from the 5' end of the T7 promoter to create a T7 promoter split at various ratios. . After transcription, the complementary sequence of the aptamer was placed on the template, and the transcription reaction of the resulting product was monitored. As shown in Figure 3, as a result of sequential insertion from 0:20 to 5:15 starting from the 5' end region, the fluorescence value rapidly decreased after the 3:17 ratio due to the absence of SP1, which was due to the T7 promoter 5' This indicates that the 3-nucleotide portion of the page is important for T7 RNA polymerase to specifically recognize the T7 promoter, and in the case of 5:15, it was confirmed that transcription hardly progresses even when both SP1 and SP2 are present. It can be seen that if there is a nick site after the 5-mer on the 5' side of the T7 promoter, T7 RNA polymerase does not recognize it normally. In order to form an intact double-stranded T7 promoter through the formation of a three-way junction structure, it was confirmed that a split ratio of 4:16 from the 5' end region was most suitable.

Therefore, in one embodiment of the present invention, the split ratio can be adjusted appropriately, and most preferably, the Ya:Yb split ratio can be 16:4.

In the probe set of the present invention, the label may be selected from the group consisting of a chemical label, an enzyme label, a radioactive label, a fluorescent label, a luminescent label, a chemiluminescent label, and a metal label.

In a preferred embodiment of the present invention, the general formula I of the first probe may be modified into general formula I', which further includes an overlapping sequence W between Y and Z;

[General formula I']

5'-X-Y-W-Z-3'

The general formula III of the second probe can be transformed into general formula III', which additionally includes an overlapping sequence W' between Yb and Z';

[Formula III']

3'-Yb-W'-Z'-5'

It may be an isothermal single reaction probe set for detecting target molecules.

The overlapping sequence was introduced to overcome the fact that when a target molecule is present, the three-way junction structure is not completely formed due to too short an overlap, so the T7 promoter is not formed and transcription does not occur effectively. Overlapping sequences W and W' are complementary to each other.

Therefore, in a preferred embodiment, specifically, in Example 1-2, structural optimization was performed to apply the T7 promoter with the Ya:Yb ratio of 16:4 to the three-way splicing construct. The 16-nucleotides on the 3' side of the T7 promoter bind complementary to a single template (Signal template, ST) and are common. Only when target RNA is present, combine the first probe consisting of ST and split T7-16 (Split T7-16, ST-16) with the second probe (Split T7-4, ST-4) to identify the nick site. Since a complete T7 promoter must be formed, the Tm value of the overlapping sequence portion is the most important factor, and the Tm value was adjusted by placing additional overlapping sequences (0 to 4 bp).

As a result, as shown in Figure 4, when the overlapping sequence (N = 0) is 4bp of the T7 promoter, no fluorescence is produced when the target sequence is not present, so it is suitable for the purpose of not forming a structure, but when the target nucleic acid sequence is present, it is suitable for the purpose of not forming a structure. In this case, it can be confirmed that the three-way structure is not completely formed due to too short overlap, so the T7 promoter is not formed and transcription is not performed effectively. It was confirmed that when more than 1 bp of overlapping sequence was added (N=1), high fluorescence values were observed only when the target sequence was present. Through this, it was confirmed that in order to stably form a 3-way junction structure and induce a transcriptional reaction, an overlap of at least 1 bp in addition to the 4 bp from the T7 promoter is required. If the added overlapping sequence exceeds 3 bp, non-specific transcription proceeds even if the target sequence is not present, so the appropriate additional overlapping sequence was set to 2 bp and the total overlapping region was set to 6 bp (4 bp from the T7 promoter). Additionally, as confirmed in Example 1-2, if they are not complementary, a bulge may be formed in the three-way bonded structure, which may reduce the stability of the structure (FIG. 5).

In one embodiment of the present invention, the length of the overlapping sequences W and W' may be 1bp or 2bp.

In the probe set of the present invention, the isothermal single reaction can be performed at a constant temperature in the range of 15°C to 50°C. The temperature in the range of 15°C to 50°C is a temperature known in the art at which enzymes act, and is not limited thereto as long as the desired effect of the present invention can be obtained. However, as confirmed in Examples 1-3 of the present invention, the reaction of the present invention was confirmed to be most efficient at 37°C (FIG. 8C). Therefore, the constant temperature of the isothermal single reaction is preferably 34°C to 40°C.

In the probe set of the present invention, the probe set can detect circular RNA separately from linear RNA. Specifically, as shown in Figure 12, the first and second probes of the present invention can each bind to both ends of linear RNA, and the first probe only binds to both ends of the linear RNA to create circular RNA. and the second probe can be combined, and the T7 promoter is completed. Therefore, using the probe set of the present invention, the T7 promoter can be formed only in the case of circular RNA, unlike linear RNA, and transcription can proceed, so it can be used to detect circular RNA.

In the probe set of the present invention, the polymerase is bacteriophage T7 RNA polymerase, bacteriophage T3 polymerase, bacteriophage RNA polymerase, bacteriophage ΦII polymerase, Salmonella bacteriophage sp6 polymerase, Pseudomonas bacteriophage gh-1 polymerase, Escherichia coli (E coli) RNA polymerase holoenzyme, E. coli RNA polymerase core enzyme, human RNA polymerase I, human RNA polymerase II, human RNA polymerase III, human mitochondrial RNA polymerase and variants thereof. It may be selected from the group consisting of, but is not limited thereto.

In one embodiment of the present invention, bacteriophage T7 RNA polymerase was used as the polymerase.

In the probe set of the present invention, the isothermal single reaction is performed simultaneously in a single reaction solution containing Tris-HCl, MgCl2, DTT, spermidine, rNTPs, RNase inhibitor, and SSB (Single-Stranded DNA Binding Protein). It can be characterized as being.

Specifically, in Examples 1-3, optimization experiments on buffer, enzyme, reaction time, and reaction temperature were performed to obtain the best performance of the proposed probe set.

In the present invention, the single reaction solution preferably contains 1 to 50mM of MgCl2, 0.1 to 10mM of DTT, 0.1 to 10mM of spermidine, 1 to 100ng/μL of single-stranded DNA binding protein (SSB), 1 to Containing 100U of T7 RNA polymerase, more preferably 1 to 30mM MgCl2, 0.5 to 5mM DTT, 1 to 5mM spermidine, 10 to 50ng/μL single stranded DNA binding protein (SSB), 10 to 50U of T7 RNA polymerase, most preferably 10mM MgCl2, 1.5mM DTT, 3mM spermidine, 20ng/μL single stranded DNA binding protein (SSB), 20U of T7 RNA polymerase. It may include (FIGS. 6 to 9).

The present invention also provides a composition for detecting a target molecule, including the isothermal single reaction probe set for detecting the target molecule.

The composition for detecting a target molecule may further include tools and/or reagents commonly used for detection and/or analysis of the target molecule in addition to nucleic acid sequences and peptides that specifically bind to the target molecule. These tools or reagents may include suitable carriers, labels capable of producing a detectable signal, solubilizers, detergents, buffers, stabilizers, etc. If the labeling substance is a fluorescent substance, it may further include a substrate necessary to measure the fluorescence intensity. Suitable carriers include soluble carriers, such as physiologically acceptable buffers known in the art (e.g., PBS, etc.); insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, polysaccharide, etc.; It may include polymers such as magnetic particles plated with metal on latex, other paper, glass, metal, agarose, and/or combinations thereof.

In the composition of the present invention, the composition may include an isothermal single reaction probe set for detecting two or more target molecules.

In the composition of the present invention, the isothermal single reaction probe sets for detecting two or more target molecules each include different interactive labeling systems; Each of the two or more probe sets binds to a different target molecule; This may enable multiple detection of different target molecules.

That is, the two or more types of targets may be different target sites present in a single pathogen. In this case, more accurate and precise diagnosis is possible by effectively detecting different sites of the target.

Specifically, in Examples 2 and 3 of the present invention, detection probes were designed to target different target sites using SARS-CoV-2 as the target material. As a result, probes targeting the two genetic loci indicated as L2 and L4 were designed, and the results showed that the two probes specifically bound to each region of the target nucleic acid sequence (see FIG. 15). Therefore, more accurate detection is possible because other parts of the target have been effectively detected.

Furthermore, the simultaneous detection of the D614G mutation and the N gene of SARS-CoV-2 in one tube was tested, where the mango aptamer and malachite green aptamer, respectively, allowed the N gene and D614G of SARS-CoV-2 to fluoresce without interference. released (Figure 17C). It was confirmed that targets for the N gene and D614G could be analyzed simultaneously up to 100 fM, enabling very sensitive detection (Figure 17D).

In addition, the two or more types of targets may be interactively different molecular diagnostic targets, for example, infectious harmful microorganisms, and in this case, simultaneous detection and diagnosis of different pathogens is possible.

Specifically, in Example 6 of the present invention, detection of two pathogenic bacteria was performed, and as a result, it was confirmed that both E. coli and MRSA specifically bound to each probe and generated high fluorescence signals. Therefore, the present invention makes it possible to detect and diagnose different pathogens individually or simultaneously.

According to another preferred embodiment of the present invention, the isothermal single reaction may be performed at a constant temperature in the range of 15°C to 50°C.

The present invention also provides a kit for detecting a target molecule, including the composition for detecting the target molecule, a polymerase, and an isothermal single reaction solution.

The kit of the present invention described above in this specification may additionally include various polynucleotide molecules, enzymes, and various buffers and reagents. Additionally, the kit of the present invention may include reagents necessary for carrying out positive control and negative control reactions. The optimal amount of reagent to be used in any one particular reaction can be readily determined by one skilled in the art upon learning the disclosure herein.

Since the kit of the present invention is manufactured to detect target molecules using the probe set of the present invention described above, duplicate content is omitted to avoid complexity of the present specification.

In addition, the present invention provides a method for detecting a target molecule using the isothermal single reaction probe set for detecting the target molecule, and may specifically include the following steps:

(a) treating the sample with an isothermal one-pot reaction probe set for detecting target molecules consisting of the first probe and the second probe of the present invention to bind or hybridize with the target molecule;

(b) treating the hybridization product of step (a) with polymerase to initiate transcription; and

(c) treating the transcription product of step (b) with an aptamer-reactive material to detect signal generation of the aptamer in the transcription product.

At this time, the signal generation in step (c) is characterized by indicating the presence of the target molecule in the sample.

The target molecule detection method of the present invention is characterized by detecting the target molecule under isothermal single reaction conditions without a separate amplification reaction.

Therefore, the method for detecting a target molecule using the probe set of the present invention is designed so that the hybridization step, transcription reaction, and aptamer signal reaction occur simultaneously, and steps (a) to (c) of the present invention are performed in one container. Can be performed simultaneously.

Additionally, the aptamer used to confirm the final signal is not limited to the malachite green aptamer, and any type of RNA-based fluorescent aptamer can be used.

In one embodiment of the present invention, an isothermal nucleic acid amplification reaction may be performed prior to the method of detecting a target molecule using the isothermal single reaction probe set for detecting the target molecule.

The type of isothermal nucleic acid amplification reaction is not particularly limited, but in one embodiment of the present invention, the isothermal nucleic acid amplification reaction may be RPA (Recombinase Polymerase Amplification).

As used herein, the term "recombinant-polymerase amplification method" refers to a technology that can confirm DNA and RNA amplification. Unlike conventional PCR, it uses a DNA-binding protein and a recombinase to quickly and accurately amplify the target sequence. It is a technology that can amplify a sequence. The RPA method uses mesophilic polymerase to enable amplification even under isothermal conditions, so it has the advantage of being able to detect and diagnose viruses in a short period of time using an isothermal device without special equipment.

It was confirmed that when RPA was performed before step (a), the signal was amplified compared to when the STAR reaction of the present invention was performed (see FIG. 12). Therefore, in connection with the present invention, isothermal nucleic acid amplification reaction can be performed as a method for more sensitive detection of target molecules.

The present invention also provides an on-site molecular diagnosis method using the isothermal single reaction probe set for detecting the target molecule.

In the specification of the present invention, “on-site molecular diagnosis method” refers to a method that allows for on-site diagnosis in medical settings and precisely diagnoses the disease by directly examining the molecular level within the cell and the genes (RNA, DNA) of the germ.

The point-of-care molecular diagnostic method may include the following steps:

(a) treating the sample with an isothermal one-pot reaction probe set for detecting a target molecule consisting of a first probe and a second probe to hybridize it with the target molecule;

(b) treating the hybridization product of step (a) with polymerase to initiate transcription; and

(c) treating the transcription product of step (b) with an aptamer-reactive material to detect signal generation of the aptamer in the transcription product.

At this time, the signal generation in step (c) is characterized by indicating the presence of the target molecule in the sample.

In the on-site molecular diagnostic method of the present invention, the molecular diagnostic method can be used to detect pathogenic microorganisms.

In the specification of the present invention, “pathogenic microorganism” refers to a microorganism that parasitizes the human body and causes disease, is not limited to the pathogenic microorganism described in the examples of the present invention, and can be applied to all pathogenic microorganisms.

In the field diagnostic method of the present invention, the pathogenic microorganisms include Staphylococcus Aureus, Vibrio vulnificus, E. coli, and Middle East Respiratory Syndrome coronavirus. Coronavirus), Influenza A virus, Severe Acute Respiratory Syndrome Coronavirus, Respiratory Syncytial Virus (RSV), Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV), Human papillomavirus (HPV), human parainfluenza virus (HPIV), dengue virus, hepatitis B virus (HBV), yellow fever virus, rabies virus, plasmodium, cytomegalovirus (CMV), mycobacterium tuberculosis, Chlamydia trachomatis, rotavirus, human metapneumovirus (hMPV), Crimean-Congo hemorrhagic fever virus, Ebola virus, Zika virus, henipavirus, norovirus, Lassa virus, rhinovirus. Viruses, flavivirus, Rift Valley fever virus, hand, foot and mouth disease virus, Salmonella sp., Shigella sp., Enterobacteriaceae sp., Pseudomonas sp., Moraxella sp.), Helicobacter sp., and Stenotrophomonas sp., but is not limited thereto.

In other words, the method for detecting target molecules using the probe set of the present invention acts as a powerful diagnostic method for detecting target molecules of interest, and not only provides short diagnosis time, high sensitivity and specificity, and simple analysis procedures, Since it does not require expensive equipment or diagnostic experts, it will be an appropriate diagnostic method for new infectious diseases that require rapid response.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

[Preparation example]

Preparation of reagents and materials

All DNA sequences were synthesized by Bionics (Seoul, Korea). All plasmids were purchased from Integrated DNA Technology (Skokie, IL, USA). T7 RNA polymerase, RNase inhibitor, T4 gene 32 protein (single-strand binding protein), and ribonucleotide (rNTP) solution were purchased from Enzynomics (Daejeon, Korea). HiScribe T7 High Yield RNA Synthesis Kit, Monarch RNA Cleanup Kit, and DNase I were purchased from New England Biolabs (Ipswich, GA, USA). Bacterial RNA kit was purchased from OMNI International (Kennesaw, NY, USA). Tris-HCl and MgCl ₂ were purchased from Biosesang (Seongnam, Korea). Dithiothreitol (DTT) was purchased from ThermoFisher Scientific (Seoul, Korea). TO1-3PEG-Biotin fluorophore (TO1-Biotin) was purchased from Applied Biological Materials (Richmond, Canada). Malachite green chloride and spermidine were purchased from Sigma-Aldrich (St. Louis, MO, USA). All experiments were performed with DEPC-treated water to prevent RNA degradation.

Preparation of DNA oligonucleotides

The sequence of the oligonucleotide used in the present invention is as follows.

DNA Probe Sequence (5'→3') sequence number Split promoter template GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC TTA AAA ATC CCG GCC AGA TTT TGC CAA TCA CCC TAT AGT GAG TCG TAT TA GTTAGA TTT TGC CAA TCA One Split promoter 1-0 TTGGCAAAATCTAAC 2 Split promoter 2-20 TAATACGACTCACTATAGGG 3 Split promoter 1-1 TTGGCAAAATCTAAC T 4 Split promoter 2-19 AATACGACTCACTATAGGG 5 Split promoter 1-2 TTGGCAAAATCTAAC TA 6 Split promoter 2-18 ATACGACTCACTATAGGG 7 Split promoter 1-3 TTGGCAAAATCTAAC TAA 8 Split promoter 2-17 TACGACTCACTATAGGG 9 Split promoter 1-4 TTGGCAAAATCTAAC TAAT 10 Split promoter 2-16 ACGACTCACTATAGGG 11 Split promoter 1-5 TTGGCAAAATCTAAC TAATA 12 Split promoter 2-15 CGACTCACTATAGGG 13 N gene of SARS-CoV-2 Split T7-16 (ST-16) - Universal ACGACTCACTATAGGG 14 Overlap 0-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TGCCAGCCATTCTAG 15 Overlap 0-ST-4 CAGCATCACCGCCAT TAAT 16 Overlap 1-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTGCCAGCCATTCTAG 17 Overlap 1-ST-4 CAGCATCACCGCCATA TAAT 18 Overlap 2-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTTGCCAGCCATTCTAG 19 Overlap 2-ST-4 CAGCATCACCGCCATAA TAAT 20 Overlap 3-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTTTGCCAGCCATTCTAG 21 Overlap 3-ST-4 CAGCATCACCGCCATAAA TAAT 22 Overlap 4-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTTTTGCCAGCCATTCTAG 23 Overlap 4-ST-4 CAGCATCACCGCCATAAAA TAAT 24 Bulge 1-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTTTGCCAGCCATTCTAG 25 Bulge 1-ST-4 CAGCATCACCGCCATTAA TAAT 26 Bulge 2-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTTTTGCCAGCCATTCTAG 27 Bulge 2-ST-4 CAGCATCACCGCCATTTAA TAAT 28 Bulge 3-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTTTTTGCCAGCCATTCTAG 29 Bulge 3-ST-4 CAGCATCACCGCCATTTTAA TAAT 30 Bulge 4-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTTTTTTGCCAGCCATTCTAG 31 Bulge 4-ST-4 CAGCATCACCGCCATTTTTAA TAAT 32 Bulge 5-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTTTTTTTGCCAGCCATTCTAG 33 Bulge 5-ST-4 CAGCATCACCGCCATTTTTTAA TAAT 34 Locus 1-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTATGAGGAACGAGAAG 35 Locus 1-ST-4 TGTTGCGACTACGTGAA TAAT 36 Locus 2-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTTGCCAGCCATTCTAG 37 Locus 2-ST-4 CAGCATCACCGCCATAA TAAT 38 Locus 3-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTGCT TTA GTG GCA GTA 39 Locus 3-ST-4 TGT GTT ACA TTG TAT TAAT 40 Locus 4-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTTGTTACATTGTATGC 41 Locus 4-ST-4 TCTGCCGAAAGCTTGAA TAAT 42 Locus 5-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTAGTTCCTGGTCCCCA 43 Locus 5-ST-4 GTTCCTTGTCTGATTAA TAAT 44 S gene (D614G Mutation) of SARS-CoV-2 Signal template-G GGATCCATTCGTTACCTGGCTCTCGCCAGTCGGGATCC CCCTATAGTGAGTCGTATTA TTAGTT G ACACCCTGAT 45 Signal template-A GGATCCATTCGTTACCTGGCTCTCGCCAGTCGGGATCC CCCTATAGTGAGTCGTATTA TTAGTT A ACACCCTGAT 46 Signal template-T GGATCCATTCGTTACCTGGCTCTCGCCAGTCGGGATCC CCCTATAGTGAGTCGTATTA TTAGTT T ACACCCTGAT 47 Signal template-C GGATCCATTCGTTACCTGGCTCTCGCCAGTCGGGATCC CCCTATAGTGAGTCGTATTA TTAGTT C ACACCCTGAT 48 D614G Mutation ST-4 CAGGGACTTCTGTGCAA TAAT 49 16S rRNA of Escherichia coli and Staphylococcus aureus E. coli L1-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTCGGGTAACGTCAATG 50 E. coli L1-ST-4 CCGGTGCTTCTTCTGAA TAAT 51 E. coli L2-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTCCACGCTTTCGCACC 52 E. coli L2-ST-4 AATCCTGTTTGCTCCAA TAAT 53 S. aureus L1-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTCGCTTTCGCACATCA 54 S. aureus L1-ST-4 CCTGTTTGATCCCCAAA TAAT 55 S. aureus L2-ST GTACGACAACTACCCCATACCAAACCTTCCTTCGTAC CCCTATAGTGAGTCGTATTA TTGGACTTAACCCAACA 56 S. aureus L2-ST-4 GTTGCGCTCGTTGCGAA TAAT 57

The underlined part indicates the mango aptamer sequence and the MG aptamer sequence, and the part in bold indicates the T7 promoter region. In the S gene (D614G Mutation) of SARS-CoV-2, the underlined, bold, and italicized parts indicate the mutation site.

DNA Probe Sequence (5'→3') sequence number SARS-CoV-2 N gene_IVT-FP TAATACGACTCACTATAGGG GCAGTCAAGCCTCTTCTCGT 58 SARS-CoV-2 N gene_IVT-RP AACATTGGCCGCAAATTGCA 59 SARS-CoV-1_IVT-FP TAATACGACTCACTATAGGG GCAGTCAAGCCTCTTCTCGC 60 SARS-CoV-1_IVT-RP GGTGTGACTTCCATGCCAAT 61 MERS-CoV_IVT-FP TAATACGACTCACTATAGGG ATAGTCAATCATCTTCAAGA 62 MERS-CoV_IVT-RP TTCTGATGGGTAAGTTTAAA 63 HCoV-229E-FP TAATACGACTCACTATAGGG GTGCTCCTTCCCGGTCTCAG 64 HCoV-229E-RP CTTCCAAGTTGTGGTCAAG 65 HCoV-NL63-FP TAATACGACTCACTATAGGG GCTCTAATAACTCATCTCGT 66 HCoV-NL63-RP TCCCCCATATTGTGATTAAA 67 HCoV-OC43-FP TAATACGACTCACTATAGGG CTGCTCCTAATTCCAGATCT 68 HCoV-OC43-RP AACTCTAATCTTGATCCAAA 69 HCoV-HKU1-FP TAATACGACTCACTATAGGG CTGCTTCTAATAGTCGACCA 70 HCoV-HKU1-RP AAGTCTAATTTAGAACCAAA 71 SARS-CoV-2 S gene (G614)_IVT-FP TAATACGACTCACTATAGGG TCTAACCAGGTTGCTGTTCTTTATCAGG G TGTTAACTGCACAGAAGTCCC 72 SARS-CoV-2 S gene (G614)_IVT-RP GGAGTAAGTTGATCTGCATGAATAGCAACAGGGACTTCTGTGCAGTTAAC 73 SARS-CoV-2 S gene (D614)_IVT-FP TAATACGACTCACTATAGGG TCTAACCAGGTTGCTGTTCTTTATCAGG A TGTTAACTGCACAGAAGTCCC 74 SARS-CoV-2 S gene (D614)_IVT-RP GGAGTAAGTTGATCTGCATGAATAGCAACAGGGACTTCTGTGCAGTTAAC 75

The part in bold indicates the T7 promoter region. In the SARS-CoV-2 S gene (G614, D614G Mutation), the underlined, bold, and italicized parts indicate the mutation site.

Preparation of target RNA sequence

Target RNA was generated through in vitro transcription. The sequence information of the target RNA used in the present invention is shown in [Table 3].

Name Sequence (5'→3') SSize (bp) Sequence number SARS-CoV-2 N gene
GCAGUCAAGCCUCUUCUCGUUCCUCAUCACGUAGUCGCAACAGUUCAAGAAAUUCAACUCCAGGCAGCAGUAGGGGAACUUCUCCUGCUAGAAUGGCUGGCAAUGGCGGUGAUGCUGCUCUUGCUUUGCUGCUGCUUGACAGAUUGAACCAGCUUGAGAGCAAAAUGUCUGGUAAAGGCCAAACAACAAGGCCAAACUGUCACUAAGAAAUCUGCUGCUGAGGCUUCUAAAGAAGCCUCGGCAAAAACGU ACUGCCACUAAAGCAUACAAUGUAACACAAGCUUUCGGCAGACGUGGUCCAGAACAAACCCAAGGAAAUUUUGGGGACCAGGAACUAAUCAGACAAGGAACUGAUUACAAACAUUGGCCGCAAAUUGCA 380 76 SARS-CoV-1
GCAGUCAAGCCUCUUCUCGCUCCUCAUCACGUAGUCGCGGUAAUUCAAGAAAUUCAACUCCUGGCAGCAGUAGGGGAAAUUCUCCUGCUCGAAUGGCUAGCGGAGGUGGUGAAACUGCCCUCGCGCUAUUGCUGCCUAGACAGAUUGAACCAGCUUGAGAGGCAAAGUUUCUGGUAAAGGCCAAACAACAAGGCCAAACUGUCACUAAGAAAUCUGCUGCUGAGGCAUCUAAAAAAGCCUCGCCAAAA ACGUACUGCCACAAAACAGUACAACGUCACUCAAGCAUUUGGGAGACGUGGUCCAGAACAAACCCAAGGAAAUUUCGGGGACCAAGACCUAAUCAGACAAGGAACUGAUUACAAACAUUGGCCGCAAAUUGCACAAUUUGCUCCAAGUGCCUCUGCAUUCUUUGGAAUGUCAGCAUUGGCAUGGAAGUCACACC 442 77 MERS-CoV AUAGUCAAUCAUCUUCAAGAGCCUCUAGCUUAAGCAGAAACUCUUCCAGAUCUAGUUCACAAGGUUCAAGAUCAGGAAACUCUACCCGCGGCACUUCUCCAGGUCCAUCUGGAAUCGGAGCAGUAGGAGGUGAUCUACUUUACCUUGAUCUUCUGAACAGACUACAAGCCCUUGAGUCUGGCAAAGUAAAGCAAUCGCAGCCAAAAGUAAUCACUAAGAAAGAUGCUGCUGCUGCUAAAAAUAAGA UGCGCCACAAGCGCACUUCCACCAAAAGUUUCAACAUGGUGCAAGCUUUUGGUCUUCGCGGACCAGGAGACCUCCAGGGAAACUUUGGUGAUCUUCAAUUGAAUAAACUCGGCACUGAGGACCCACGUUGGCCCCAAAUUGCUGAGCUUGCUCCUACAGCCAGUGCUUUUAUGGGUAUGUCGCAAUUUAAACUUACCCAUCAGAA 451 78 HCoV-229E GUGCUCCUUCCCGGUCUCAGUCGAGGUCGCAGAGUCGCGGUCGUGGUGAAUCCAAACCUCAAUCUCGGAAUCCUUCAAGUGACAGAAACCAUAACAGUCAGGAUGACAUCAUGAAGGCAGUUGCUGCGGCUCUUAAAUCUUUAGGUUUUGACAAGCCUCAGGAAAAAGAUAAAAAGUCAGCGAAAACGGGUACUCCUAAGCCUUCUCGUAAUCAGAGUCCUGCUUCUUCUCAAACUUCUGC CAAGAGUCUUGCUCGUUCUCAGAGUUCUGAAACAAAAGAACAAAAGCAUGAAAUGCAAAAGCCACGGUGGAAAAGACAGCCUAAUGAUGAUGUGACAUCUAAUGUCACACACAUGUUUUGGCCCCAGAGACCUUGACCACAACUUUGGAAG 391 79 HCoV-NL63 GCUCUAAUAACUCAUCUCGUGCUAGCAGUCGUUCUUCAACUCGUAACAACUCACGAGACUCUUCUCGUAGCACUUCAAGACAACAGUCUCGCACUCGUUCUGAUUCUAACCAGUCUUCUUCAGAUCUUGUUGCUGCUGUUACUUUGGCUUUAAAGAACUUAGGUUUUGAUAACCAGUCGAAGUCACCUAGUUCUUCUGGUACUUCCACUCCUAAGCCUAAUAAGCCU CUUUCUCAACCCAGGGCUGAUAAGCCUUCUCAGUUGAAGAAACCUCGUUGGAAGCGUGUUCCUACCAGAGAGGAAAAUGUUAUUCAGUGCUUUGGUCCUCGUGAUUUUAAUCACAAUAUGGGGGA 355 80 HCoV-OC43 CUGCUCCUAAUUCCAGAUCUACUUCGCGCACAUCCAGCAGAGCCUCUAGUGCAGGAUCGCGUAGUAGAGCCAAUUCUGGCAAUAGAACCCCUACCUCUGGUGUAACACCUGACAUGGCUGAUCAAAUUGCUAGUCUUGUUCUGGCAAAACUUGGCAAGGAUGCCACUAAACCUCAGCAAGUAACUAAGCAUACUGCCAAAGAAGUCAGACAGAAAAUUUUGAAUAAGCCCCGCCAGAAGAGGAGCC CCAAUAAACAAUGCACUGUUCAGCAGUGUUUUGGUAAGAGAGGCCCUAAUCAGAAUUUUGGUGGUGGAGAAAUGUUAAAACUUGGAACUAGUGACCCACAGUUCCCCAUUCUUGCAGAACUCGCACCCACAGCUGGUGCGUUUUUCUUUGGAUCAAGAUUAGAGUU 412 81 HCoV-HKU1 CUGCUUCUAAUAGUCGACCAGGUUCACGUUCUCAAUCACGUGGACCCAAUAAUCGUUCAUUAAGUAGAAGUAAUUCUAAUUUUAGACAUUCAGAUUCUAUAGUAAAACCUGAUAUGGCUGAUGAGAUCGCUAAUCUUGUUUUAGCCAAGCUUGGUAAAGAUUCUAAACCUCAGCAAGUCACUAAGCAAAAUGCCAAGGAAAUCAGGCAUAAAAUUUUAACAAAACCUCGCCAAAAGCGAACUCCU AAUAAACAUUGUAAUGUUCAACAGUGUUUUGGUAAAAGAGGACCUUCUCAAAAUUUUGGUAAUGCUGAAAUGUUAAAGCUUGGUACUAAUGAUCCUCAGUUUCCUAUUCUUGCAGAAUUAGCUCCUACACCAGGUGCUUUUUUCUUUGGUUCUAAAUUAGACUU 409 82 SARS-CoV-2 S gene (G614) UCUAACCAGGUUGCUGUUCUUUAUCAGGGUGUUAACUGCACAGAAGUCCCUGUUGCUAUUCAUGCAGAUCAACUUACUCC 80 83 SARS-CoV-2 S gene (D614) UCUAACCAGGUUGCUGUUCUUUAUCAGGAUGUUAACUGCACAGAAGUCCCUGUUGCUAUUCAUGCAGAUCAACUUACUCC 80 84 E. coli 16S rRNA AGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCNGCGUGUGUAUGAAGAAGGCCUUCGGGUUGUAAAGUACUUUCAGCGGGGAGGAAGGGAGUAAAGUUAAUACCUUUGCUCAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACG CAGGCGGUUUGUUAAGUCAGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUCUGAUACUGGCAAGCUUGAGUCUCGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACGAAGACUGACGCUCAGGUGCGAAAGCGUGGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCUC GACUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGANNUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCGCACAAGCGGUGGA 610
(327-936) 85 S. aureus 16S rRNA CAGAAGAGGAAAGUGGAAUUCCAUGUGUAGCGGUGAAAUGCGCAGAGAUAUGGAGGAACACCAGUGGCGAAGGCGACUUUCUGGUCUGUAACUGACGCUGAUGUGCGAAAGCGUGGGGAUCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGAGUGCUAAGUGUUAGGGGGUUUCCCGCCCCUUAGUGCUGCAGCUAACGCAUUAAGCACUCCGCCUGGGGAGUACGACCGCAA GGUUGAAACUCAAAGGAAUUGACGGGGACCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAAGCAACGCGAAGAACCUUACCAAAUCUUGACAUCCUUUGACAACUCUAGAGAUAGAGCCUUCCCCUUCGGGGGACAAAGUGACAGGUGGUGCAUGGUUGUCGUCAGCUCGUGUGUCGUGAGAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAAGCUUAGUUGCCAUCAUUAAGU UGGGCACUCUAAGUUGACUGCCGGUGAGACAAACCGGAGGA 524
(666-1188) 86

Optimization of Split T7 promoter-based DNA probe and reaction conditions

1-1. T7 promoter split ratio

Since the core of the present invention is the effective formation of a split T7 promoter in the presence of target RNA by inducing a three-way splicing structure, first, prior to the present invention, the existing T7 promoter was split and the transcription efficiency according to each ratio was analyzed. (Figure 2).

Transcription reaction using three types of DNA probes (Split Promoter 1 (SP1), Split Promoter 2 (SP2), and Template (T)) to confirm transcription with the split T7 promoter. was carried out. The reaction mixture contained 2 μL of SP1, SP2, and T (1 μM each), 2 μL 10x T7 buffer, 0.8 μL T7 RNA polymerase (2 U/μL), 0.4 μL RNase inhibitor (0.08 U/μL), and 2 μL TO1. -Consisted of biotin (1 μM), 1 μL rNTPs (2.5 mM each), and 11.8 μL DEPC treated water. The reaction mixture was incubated at 37°C for 1 hour and then transferred to a 384-well plate. Fluorescence signals were measured at excitation and emission wavelengths of 507 nm and 547 nm, respectively, using a microplate reader (SpectraMax iD5 Multi-Mode Microplate Reader, Molecular Devices, USA).

Target RNA was generated through in vitro transcription . Sequence information of the target RNA used in the present invention is as shown in [Table 3] above.

Specifically, each plasmid was used as a PCR template, and a DNA sequence corresponding to the target RNA region was first generated using primers containing the T7 promoter sequence. Next, a transcription reaction solution consisting of 1 μg of template DNA, 1.5 μL of 10X reaction buffer (final 0.75×), 1.5 μL of rNTP (7.5 mM each), 1.5 μL of T7 RNA polymerase Mix, and 14.5 μL of DEPC-treated water was prepared. Then, the transcription reaction was performed by incubation at 37°C for 16 hours, and then the PCR product was digested with DNaseⅠ. RNA transcription products were purified using the Monarch RNA Cleanup Kit, quantified by nanodrop at a wavelength of 260 nm, and stored at -80°C before use.

To control the formation of the T7 promoter, nick sites were sequentially inserted starting from the 5' end of the T7 promoter to create a T7 promoter split at various ratios. After transcription by placing the complementary sequence of the mango aptamer on the template, the transcription reaction was monitored by measuring the fluorescence signal of TO-1 biotin. As the sequence of the split T7 promoter DNA probe, sequences of SEQ ID NOs: 1 to 13 in [Table 1] described above were used.

As a result, as shown in Figure 3, addition of T+SP2 or T+SP1+SP2 in the intact T7 promoter (0:20 split ratio) resulted in efficient transcription as indicated by high fluorescence signal. Using a split T7 promoter at a 1:19 ratio resulted in increased signal compared to the intact T7 promoter even in the presence of a nick site (T+SP1+SP2). Additionally, transcription occurred efficiently in the presence of T+SP2. When the split ratio was changed from 2:18 to 4:16, transcription occurred efficiently in the presence of T+SP1+SP2. After the 3:17 ratio, the fluorescence value decreased dramatically due to the absence of SP1, indicating that the 3-nucleotide region on the 5' side of the T7 promoter is important for T7 RNA polymerase to specifically recognize the T7 promoter. You can. In addition, in the case of 5:15, it was confirmed that transcription hardly progresses even when both SP1 and SP2 are present. From this, it can be seen that if a nick site exists after the 5-mer on the 5' side of the T7 promoter, T7 RNA polymerase operates normally. It can be seen that this means that it is not recognized.

It was confirmed that the 4:16 split ratio was most suitable to form an intact double-stranded T7 promoter through the formation of a 3-way junction structure.

1-2. Optimization of overlapping sequences

Afterwards, structural optimization was performed to apply the T7 promoter at a 4:16 ratio to the 3-way junction construct (Figure 4).

The 16-nucleotides on the 3' side of the T7 promoter bind complementary to the signal template and are common. Since the first probe consisting of ST and ST-16 and the second probe (ST-4) should be combined to form a complete T7 promoter with a nick site only when the target RNA is present, the Tm value of the overlapping region is the most important factor. The Tm value was adjusted by placing additional overlapping sequences (0 to 4 bp). The additional overlapping sequence portion basically includes 4 bp of the 5' portion of the T7 promoter.

The sequences of SEQ ID NOs: 13 to 24 in [Table 1] described above were used as the DNA probe sequence for optimizing the overlapping sequence.

First, when the overlapping sequence (N=0) was 4bp of the T7 promoter, no fluorescence was produced when the target sequence was not present, making it suitable for the purpose of not forming a structure. However, when the target is present, it can be confirmed that the three-way junction structure is not completely formed due to too short overlap, so the T7 promoter is not formed and transcription is not performed effectively. It was confirmed that when more than 1 bp of overlapping sequence was added (N=1), high fluorescence values were observed only when the target sequence was present. Through this, it was confirmed that in order to stably form a three-way junction structure and induce a transcriptional reaction, an overlap of at least 1 bp in addition to the 4 bp from the T7 promoter is required. If the added overlapping sequence exceeded 3bp, non-specific transcription proceeded even if the target sequence did not exist.

Therefore, the appropriate additional overlapping sequence was set to 2 bp, and the total overlapping region was set to 6 bp (4 bp from the T7 promoter).

Afterwards, a bulge structure was added to the 3-way junction structure to test the stability of the T7 promoter with a double-stranded nick site (Figure 5). The DNA sequences of ST and ST-4 in which the bulge structure was introduced were the sequences of SEQ ID NOs: 25 to 34 in [Table 1] described above.

As a result, as shown in Figure 5, it was confirmed that when a bulge is added, the stability of the structure decreases and the three-way structure is not properly formed even when the target nucleic acid sequence is present, resulting in less transcription. Therefore, it was confirmed that the optimal structure is that no overlapped sequences are added other than the complementary 2 bp and no bulge structure is created.

1-3. Optimization of reaction conditions

To obtain the best performance of the proposed STAR, optimization experiments on buffer, enzyme, reaction time, and reaction temperature were performed.

The concentration of DTT was set to 0.375mM to 1.5mM and the concentration of MgCl ₂ was set to 0 to 20mM, and each case (25 types; 5X5) was tested three times to measure the fluorescence intensity. The fluorescence intensity of spermidine was measured with and without target at a concentration of 0 to 5mM, and the fluorescence intensity of single-stranded DNA binding protein (SSB) was also measured at 0 to 50 ng/ul. T7 RNA polymerase was measured at 0 to 80U, reaction time was 0 to 120 minutes, and reaction temperature was 25, 30, 37, and 41 degrees. Additionally, in order to optimize the annealing method, fast cooling, slow cooling, and no cooling (w/o cooling) were compared.

As a result, as shown in Figures 6 to 9, the ideal conditions were as follows: 10mM MgCl ₂ , 1.5mM DTT (Figure 6), 3mM spermidine, 20ng/μL single-stranded DNA binding protein (SSB) ) (Figure 7), 20U of T7 RNA polymerase, reaction time 30 minutes, reaction temperature 37°C (Figure 8), no separate cooling.

That is, due to the optimized amount of spermidine and SSB that can mediate efficient hybridization of the probe and target RNA, a three-way junction structure is effectively formed, conditions that can induce a transcription reaction without the initial heat denaturation and cooling steps. was confirmed. Therefore, it is very advantageous in terms of practical field application, considering the need to perform the entire analysis work under isothermal conditions (37°C).

1-4. Comparison of transcription efficiency for 3-way junction structures and linear structures

Finally, to compare the optimized buffer and three-way junction structure with the existing T7 promoter, real-time fluorescence monitoring was performed using SYBR Green II, which emits a high fluorescence signal by binding to single-stranded RNA.

As a result, as shown in Figure 9, there was no significant difference compared to the existing T7 promoter, and rather, it was found that transcription of the three-way junction structure had a transcription efficiency almost similar to transcription of the linear structure.

1-5. Performing gel electrophoresis to confirm the three-way junction structure

The present invention is a technology that combines a split T7 promoter based on a three-way splicing structure and fluorescent RNA aptamer technology, and a fluorescent signal is amplified when a target nucleic acid biomarker is present.

DNA probes are composed of two types of single-stranded DNA. A single template (Signal template, ST) consists of a sequence complementary to the mango aptamer, one of the fluorescent RNA aptamers (yellow), a T7 promoter sequence (blue), and a sequence complementary to half of the target sequence (black). Split T7-16 (ST-16) is a partial sequence (16mer; blue) of the T7 promoter and is complementary to the T7 promoter of ST. The Tm value is 56.3℃, which is universal because it binds complementary to ST regardless of the target material in a reaction at 37℃. Gel electrophoresis was performed to confirm the three-way junction structure, and Figure 10 shows the results of polyacrylamide gel electrophoresis.

The formation of the three-way junction structure was confirmed by PAGE (Poly Acrylamide Gel Electrophoresis) using an 8% polyacrylamide gel. Samples were incubated with 1 μM signal template (ST), 1 μM Split T7-16 (ST-16), 1 μM Split T7-4 (ST-4), 500 nM N gene RNA, and 0.5 U/μL T7 RNA polymerase at 37°C. It was prepared by incubating for a minute. Prepared samples were loaded onto the gel and loaded with 1X TBE buffer at 200 V for 40 min. Additionally, agarose gel electrophoresis was performed using a 2% agarose gel in 1X TBE buffer at 135V for 1 hour. Results were visualized using GreenStar Nucleic Acid staining solution (Bioneer, Daejeon, Korea) and gels were scanned using ChemiDoc (Bio-rad, CA, USA).

Split T7-4 (ST-4) consists of a partial sequence of the T7 promoter (4mer; blue) and the other half of the sequence capable of complementary binding to the target sequence (black). All reactions in the present invention are carried out at 37°C. The 5'-ATTATT-3' sequence of ST is complementary to ST-4, but since the Tm value is 12℃, it was designed to prevent binding at 37℃. Therefore, if the target material is not present, the ST and ST-4 probes among the DNA probes cannot bind, and thus a three-way junction structure cannot be formed, preventing the formation of a complete double-stranded T7 promoter. When a target substance is present, the target RNA preferentially binds to the complementary sequences of the target substance, ST and ST-4, to form a three-way junction structure. Through this, a double-stranded T7 promoter with a nick site is formed.

As a result, as shown in Figure 9, the formation of a three-way junction structure was confirmed in the presence of the target and T7 RNA polymerase-catalyzed transcription reaction through PAGE analysis. As shown in Figure 9, ST and ST-16 hybridized with each other (a), and ST, ST-16, and ST-4 did not form a three-way junction structure in the absence of the target (d). Additionally, when all DNA probes and targets were present (g), the presence of T7 RNA polymerase formed a three-way junction structure that generated a large amount of light-up RNA aptamers through transcription (h).

1-6. Evaluation of fluorescence emission spectra under various reaction conditions

Next, the fluorescence spectrum without each element of STAR was evaluated (Figure 11). RNA transcription occurs by T7 RNA polymerase and mango aptamer is generated. Finally, it combines with To1-biotin, a fluorescent dye that can bind to the mango aptamer, to produce a high fluorescence signal. Without one of the DNA probes (ST, ST-16, ST-4) or the target sequence, the double-stranded T7 promoter containing the nick site essential for RNA transcription is not formed and the Mango aptamer is not produced. Therefore, binding between the mango aptamer and TO1-biotin does not occur, and a negligible fluorescence signal appears. Additionally, no fluorescence signal appears even in the absence of TO1-biotin or T7 RNA polymerase. A high fluorescence signal is generated only when all elements are present (w/all), indicating that all elements of STAR each play an important role in one-pot detection of target RNA.

Measurement of detection sensitivity for each locus of SARS-CoV-2 N gene

In the present invention, it was assumed that the formation efficiency of the three-way junction structure, which is the core of STAR, may be different for each target region and lead to different detection sensitivities. SARS-CoV-2 was selected as the target material, and five types of probes were designed for certain regions of the N gene (Nucleotide positions 28809-29188) (Figure 14A). For the sequence of the N gene, refer to NCBI Accession number NC_045512. For specific sequence information about the five types of probes, the sequences of SEQ ID NOs: 35 to 44 in [Table 1] described above were used.

After confirming that each region is specific to SARS-CoV-2 through multiple sequence alignment of DNA STAR, the presence of target (F) fluorescent signal ranges from 100pM (6ⅹ10 ⁷ copies/μL) to 100fM (6 ⅹ10 ⁴ copies/μL). Fold change was calculated by dividing by the absence of target (FNTC; non-target control). As shown in the heatmap in Figure 14B, transcription efficiency was dependent on the targeting locus. Among the five DNA probe sets (Locus 1-5), the DNA probes for L2 and L4 showed higher fold changes than the other probes (L1, L3, and L5) and determined the target N gene up to 100 fM (Figure 14C). As a result, it was confirmed that the sensitivity was different for each region, and it was confirmed that among the five regions, L2 and L4 could be quantitatively detected up to 100fM.

Targeting two loci to improve detection sensitivity of STAR

In order to improve sensitivity based on the experimental results for each region (FIG. 14), a method of using two probes simultaneously was introduced by targeting various regions of the target gene (N gene) in one reaction. Based on the results of Example 2, the STAR probe targeting L2 and L4, which had the best sensitivity, was selected. Figure 15A shows a schematic diagram of the SARS-CoV-2 N gene and two other loci of other human coronaviruses, with nucleotides from SARS-CoV-2 and other viral RNA sequences shown in gray. The sequence information of each DNA probe is as shown in [Table 2], and the sequence information of the target RNA is as shown in [Table 3].

Each STAR probe generates a signal by binding to each locus in the target RNA region, so the signal can be amplified. To confirm that the STAR probes in both regions (L2 and L4) simultaneously bind to one target, agarose gel electrophoresis was first performed as described above in Examples 1-5. The concentration of the L2 and L4 probe sets is 500 nM each (a and b), and when the L2 and L4 probes are present simultaneously, the concentration of each probe set is 250 nM (c). As a result, as shown in Figure 15B, when two probe sets are present simultaneously (c), the electrophoretic mobility is reduced compared to when only a single probe set (L2 or L4) is present (a and b), which results in a higher band position. confirmed by . These results demonstrate that the two STAR probes specifically bind to each region of the target RNA.

All reagents for STAR were premixed for detection of target RNA; 4 μl 10 4 (2 μM)), 4 μL TO1-biotin (1 μM) or 2 μL malachite green chloride (100 μM)), 4 μL SSB (20 ng/μL), 4 μL rNTP (2.5 mM each), 0.4 μL T7 RNA polymerase (0.5 U/μl), 0.8 μL RNase inhibitor (0.08 U/μL), and DEPC-water (maximum 36 μL). The mixing order of the reagents can be mixed arbitrarily until the target RNA is added. Next, 4 μL of target RNA was added and the reaction mixture was incubated at 37°C for 30 minutes. For dual targeting at the N gene (Figure 15A), 50 nM of each STAR probe set (L2 and L4) was added. Finally, the reaction mixture was transferred to a 384-well plate, where the fluorescence signal corresponding to TO1-biotin binding to the mango aptamer was analyzed using a microplate reader (SpectraMax iD5 Multi-Mode Microplate Reader, Molecular Devices, USA), respectively. Measurements were made at excitation and emission wavelengths of 507 nm and 547 nm. To measure the fluorescence signal from malachite green binding to the malachite green aptamer, 616 nm and 665 nm were used as excitation and emission wavelengths.

When two probe sets were used simultaneously, the fluorescence intensity was compared with when only one probe set was used. Fluorescence signals were measured in the presence of L2 probe set (100 nM), L4 probe set (100 nM), separately, and both L2 and L4 probe sets (50 nM + 50 nM), and the concentration of target N gene RNA was 1 nM. It was confirmed that the fluorescence signal increased when two probe sets were used simultaneously (L2+L4) (Figure 15C), indicating that it can be used as a good method for signal improvement, especially in limited situations where the target RNA concentration is low. .

The detection limit was measured using the STAR probe set combination at L2 and L4. The concentration of STAR probe set is 50 nM at each different locus. It was shown that target RNA can be determined up to 10 ² copies/μL by targeting two loci, which is a 600-fold improvement compared to targeting one locus, which is useful for screening SARS-CoV-2 in actual clinical situations. It shows that it is sufficient (Figure 15D).

In addition, the specificity of STAR was assessed with a DNA probe designed not to bind to six human coronaviruses, including other alphacoronaviruses and betacoronaviruses. The SARS-CoV-2 region and the nucleotides of six other coronaviruses are shown in gray (Figure 15A). In the presence of six coronavirus RNAs at a concentration of 100 nM, the fluorescence signal was similar to that observed for NTC, and a highly enhanced fluorescence signal was generated only for the SARS-CoV-2 N gene at 1 nM (Figure 15E). This indicates that only the targeted SARS-CoV-2 can be detected specifically and sensitively even in situations where the RNA target concentration is small.

4-1. Multiplex STAR for detection of SARS-CoV-2 D614G mutation

An experiment was conducted to demonstrate the versatile applicability of STAR by selecting the D614G mutation of SARS-CoV-2 as a target. In addition, multiple detection for simultaneous detection of the D614G mutation and N gene of SARS-CoV-2 in a single tube was confirmed.

The D614G mutation is known to exist in all SARS-CoV-2 variants, including alpha, beta, and delta. In Figure 16A, mutations are indicated in yellow, and mismatch sequences indicated in red were added to avoid false positive signals in wild targets. As adenine (A) is replaced by guanine (G) at position 23403 of SARS-CoV-2 genomic RNA, a specific STAR probe was rationally designed to identify this change, and malachite green aptamer and malachite green aptamer were used for multiplexing experiments. A fluorogenic dye (malachite green) with a different wavelength was used (Figure 16A). As the sequence of the DNA probe, sequences of SEQ ID NOs: 45 to 49 in [Table 1] described above were used.

Multiplex assays were performed using 4 μL 10xSTAR buffer (400mM Tris-HCl, 100mM MgCl2, 15mM DTT, 30mM spermidine), 3μL L2 probe set (2μM each), 3μL L4 probe set (2μM each), 6μL D614G mutant probe set (2μM each). ), 4 μL TO1-biotin (1 μM), 2 μL malachite green chloride (100 μM), 4 μL SSB (20 ng/μL), 4 μL rNTPs (2.5 mM each), 0.4 μL T7 RNA polymerase (0.5 U/μL), 0.8 μL RNase. Inhibitor (0.08 U/μL) and 8 μL of target RNA were added to DEPC-water (maximum 32 μL).

Different nucleobases (A, T, G, and C) at position 4 (N4) were evaluated to find the best one that gave effective discrimination of the mutant (D614G) from the wild type, resulting in The results in Figure 16B showed that G at position N4 produced high fluorescence signal in the presence of the mutant target (D614G) while minimizing background noise in the presence of the wild target. As the DNA probe sequence, sequences of SEQ ID NOs: 72 to 75 of the aforementioned [Table 2] were used, and SEQ ID NOs: 83 and 84 of the aforementioned [Table 3] were used as the target RNA sequence information.

As a result, a detection limit of 100 fM was obtained for the mutation, and it was confirmed that the wild type did not interfere with mutation detection (Figure 16C). Additionally, mixtures of mutant/wild targets with different molar ratios (0%, 0.1%, 1%, 10%, 50%, and 100%) were analyzed using the proposed STAR.

Additionally, Figure 16D shows that 0.1% of the mutant targets are distinct from wild targets, which is similar to or better than other methods, confirming the excellent specificity of the present invention.

5-2. System for multiple detection

A multiplexed STAR was demonstrated for simultaneous detection of the D614G mutation and N gene of SARS-CoV-2 in one tube, where the mango aptamer and malachite green aptamer correspond to the N gene and D614G of SARS-CoV-2, respectively. Figure 17A shows a schematic diagram of a one-pot multiplex STAR for simultaneous detection of SARS-CoV-2 N gene and D614G mutation.

Since mango and malachite green aptamers were used for simultaneous detection, we wanted to check whether the two aptamers emit fluorescence without interference. As a result, Figure 17B shows the normalized fluorescence emission spectra of TO1-biotin and MG binding to mango aptamer and MG aptamer, respectively. The fluorescence emission spectra of TO1-biotin were recorded at 520 nm to 570 nm and an excitation wavelength of 480 nm, and those of malachite green were recorded at 640 nm to 720 nm and an excitation wavelength of 600 nm. It has been done. It was confirmed that TO1-biotin and malachite green generated distinct fluorescence emission spectra (green and red) without interference after binding to the mango and malachite green aptamers, respectively (Figure 17B).

Additionally, the STAR probe for the N gene produced a highly enhanced fluorescence signal (green) through the production of the mango aptamer only when the N gene of SARS-CoV-2 was present, and the STAR probe for the D614G mutation resulted in highly enhanced fluorescence. Generated a signal. Figure 17C shows a heatmap showing the results of one-pot multiplex detection of the N gene and D614G mutation. The concentration of target RNA was 10 nM, and only when the D614G mutation was present, a malachite green aptamer was formed and a fluorescent signal (red) was generated, demonstrating the possibility of multiplex analysis (Figure 17C).

The sensitivity to both the N gene and D614G of SARS-CoV-2 was evaluated in a multiplex experiment in which the STAR probe for the N gene and the STAR probe for D614G were simultaneously present. It was confirmed that targets for the N gene and D614G could be analyzed simultaneously up to 100 fM (Figure 17D).

Therefore, it was confirmed that multiple detection of different target nucleic acid sequences can be performed sensitively and without interference with the STAR probe of the present invention.

System for pathogen detection

To determine whether the STAR assay can be used for a variety of applications, an experiment was performed to detect two pathogenic bacteria, E. coli (Escherichia coli) and S. aureus (Staphylococcus Aureus) (Figure 18). For sensitive and specific detection, 16S rRNA, which exists in large quantities in pathogens, was selected as a target, sequenced in other bacterial genera, and STAR probes specific for E. coli and S. aureus were designed.

The target RNA sequence used was the sequence of SEQ ID NOs: 85 and 86 in [Table 3], and the sequence information of the DNA probe used was the sequence of SEQ ID NO: 50 to 57 in [Table 1].

The experiment was conducted in two ways. One method is to detect total RNA extracted from pathogens, and the other method is to detect total RNA by performing a direct experiment in which analysis is performed immediately after heat treatment of the pathogen without an additional nucleic acid extraction process (Figure 17A).

Among the two experimental methods, the method of extracting nucleic acids proceeds as follows. E. coli and S. aureus were first cultured in Luria-Bertani liquid medium at 37°C for 24 hours with shaking (150 rpm). Then, 1 ml medium was extracted for total RNA using a bacterial RNA extraction kit.

In the case of the direct method performed without additional nucleic acid extraction process, the bacterial pellet was resuspended in DEPC-treated water after centrifugation at 5000 rpm and dissolved by heating at 95°C for 5 minutes. 4 μL of lysate was used for STAR analysis according to the procedure described above.

As shown in Figure 18B, it was confirmed that both E. coli and MRSA specifically bound to each probe, generating a high fluorescence signal. The extracted total RNA was quantitatively measured at a concentration ranging from 100pg/μL to 100fg/μL, and it was confirmed that detection was possible even at a concentration of 100fg/μL.

Additionally, direct STAR was performed using pathogens at a concentration of 10 ³ cells/μL. Lysates of each pathogen (E. coli and S. aureus) were prepared by heating at 95°C for 5 minutes, and then STAR was performed with E. coli and S. aureus specific STAR probes. Figure 18C shows a heatmap showing the results of direct STAR for detection of pathogenic bacteria. The concentration of each pathogen is 10 ³ cells/μL.

As a result, the method of the present invention can be used to detect various biomarkers of pathogenic bacteria as well as viruses, and can be detected directly by heat treatment without pretreatment. Therefore, when using the present invention, multiple detection and direct systems can be constructed with only one enzyme in one tube and can be used to detect pathogenic bacteria simply and quickly.

SEQUENCE LISTING <110> KONKUK UNIVERSITY INDUSTRIAL COOPERATION CORP <120> Probe Set for Isothermal One-Pot Reaction using Split T7 promoter and Uses Thereof <130> 1069767 <160> 86 <170> PatentIn version 3.2 <210> 1 <211> 105 <212> DNA <213> Artificial <220> <223> Split promoter template <400> 1 gtacgacaac taccccatac caaaccttcc ttcgtactta aaaatcccgg ccagattttg 60 ccaatcaccc tatagtgagt cgtattagtt agattttgcc aatca 105 <210> 2 <211> <212> DNA <213> Artificial <220> <223> Split promoter 1-0 <400> 2 ttggcaaaat ctaac 15 <210> 3 <211> 20 <212> DNA <213> Artificial <220> <223> Split promoter 2-20 <400> 3 taatacgact cactataggg 20 <210> 4 <211> 16 <212> DNA <213> Artificial <220> <223> Split promoter 1-1 <400> 4 ttggcaaaat ctaact 16 <210> 5 <211> 19 <212> DNA <213 > Artificial <220> <223> Split promoter 2-19 <400> 5 aatacgactc actataggg 19 <210> 6 <211> 17 <212> DNA <213> Artificial <220> <223> Split promoter 1-2 <400> 6 ttggcaaaat ctaacta 17 <210> 7 <211> 18 <212> DNA <213> Artificial <220> <223> Split promoter 2-18 <400> 7 atacgactca ctataggg 18 <210> 8 <211> 18 <212> DNA <213> Artificial <220> <223> Split promoter 1-3 <400> 8 ttggcaaaat ctaactaa 18 <210> 9 <211> 17 <212> DNA <213> Artificial <220> <223> Split promoter 2-17 < 400> 9 tacgactcac tataggg 17 <210> 10 <211> 19 <212> DNA <213> Artificial <220> <223> Split promoter 1-4 <400> 10 ttggcaaaat ctaactaat 19 <210> 11 <211> 16 <212 > DNA <213> Artificial <220> <223> Split promoter 2-16 <400> 11 acgactcact ataggg 16 <210> 12 <211> 20 <212> DNA <213> Artificial <220> <223> Split promoter 1- 5 <400> 12 ttggcaaaat ctaactaata 20 <210> 13 <211> 15 <212> DNA <213> Artificial <220> <223> Split promoter 2-15 <400> 13 cgactcacta taggg 15 <210> 14 <211> 16 <212> DNA <213> Artificial <220> <223> Split T7-16 (ST-16)-Universal <400> 14 acgactcact ataggg 16 <210> 15 <211> 72 <212> DNA <213> Artificial <220 > <223> Overlap 0-ST <400> 15 gtacgacaac tacccatac caaaccttcc ttcgtacccc tatagtgagt cgtattatgc 60 cagccattct ag 72 <210> 16 <211> 19 <212> DNA <213> Artificial <220> <223> Overlap 0-ST-4 <400> 16 cagcatcacc gccattaat 19 <210> 17 <211> 73 <212> DNA <213> Artificial <220> <223> Overlap 1-ST <400> 17 gtacgacaac taccccatac caaaccttcc ttcgtacccc tatagtgagt cgtattattg 60 ccagccattc tag 73 <210> 18 <211> 20 <212> DNA <213> Artificial <220> <223> Overlap 1-ST-4 <400> 18 cagcatcacc gccatataat 20 <210> 19 <211> 74 <212> DNA <213> Artificial <220 > <223> Overlap 2-ST <400> 19 gtacgacaac tacccatac caaaccttcc ttcgtacccc tatagtgagt cgtattattt 60 gccagccatt ctag 74 <210> 20 <211> 21 <212> DNA <213> Artificial <220> <223> Overlap 2-ST-4 <400> 20 cagcatcacc gccataataa t 21 <210> 21 <211> 75 <212> DNA <213> Artificial <220> <223> Overlap 3-ST <400> 21 gtacgacaac taccccatac caaaccttcc ttcgtacccc tatagtgagt cgtattattt 60 tgccagccat tc tag 75 <210 > 22 <211> 22 <212> DNA <213> Artificial <220> <223> Overlap 3-ST-4 <400> 22 cagcatcacc gccataaata at 22 <210> 23 <211> 76 <212> DNA <213> Artificial <220> <223> Overlap 4-ST <400> 23 13> Artificial <220> <223> Overlap 4-ST -4 <400> 24 cagcatcacc gccataaaat aat 23 <210> 25 <211> 75 <212> DNA <213> Artificial <220> <223> Bulge 1-ST <400> 25 gtacgacaac taccccatac caaaccttcc ttcgtacccc tatagtgagt cgtattattt 60 tgccagc cat tctag 75 <210> 26 <211> 22 <212> DNA <213> Artificial <220> <223> Bulge 1-ST-4 <400> 26 cagcatcacc gccattaata at 22 <210> 27 <211> 76 <212> DNA <213 > Artificial <220> <223> Bulge 2-ST <400> 27 gtacgacaac taccccatac caaaccttcc ttcgtacccc tatagtgagt cgtattattt 60 ttgccagcca ttctag 76 <210> 28 <211> 23 <212> DNA <213> Artificial <220> <223> 2 -ST-4 <400> 28 cagcatcacc gccatttaat aat 23 <210> 29 <211> 77 <212> DNA <213> Artificial <220> <223> Bulge 3-ST <400> 29 gtacgacaac taccccatac caaaccttcc ttcgtacccc tatagtgagt cgtattattt 60 gccagcc attctag 77 <210> 30 <211> 24 <212> DNA <213> Artificial <220> <223> Bulge 3-ST-4 <400> 30 cagcatcacc gccattttaa taat 24 <210> 31 <211> 78 <212> DNA <213> Artificial <220> <223> Bulge 4-ST <400> 31 gtacgacaac taccccatac caaaccttcc ttcgtacccc tatagtgagt cgtattattt 60 ttttgccagc cattctag 78 <210> 32 <211> 25 <212> DNA <220> Artificial <220> 23> Bulge 4-ST-4 <400> 32 cagcatcacc gccattttta ataat 25 <210> 33 <211> 79 <212> DNA <213> Artificial <220> <223> Bulge 5-ST <400> 33 gtacgacaac taccccatac caaaccttcc ttcgtacccc tatagtgagt cgtattattt 60 tttttgccag ccattctag 79 <210> 34 <211> 26 <212> DNA <213> Artificial <220> <223> Bulge 5-ST-4 <400> 34 cagcatcacc gccatttttt aataat 26 <210> 35 <211> 74 <212 > DNA <213> Artificial <220> <223> Locus 1-ST <400> 35 gtacgacaac taccccatac caaaccttcc ttcgtacccc tatagtgagt cgtattatta 60 tgaggaacga gaag 74 <210> 36 <211> 21 <212> DNA <213> Artificial <220> < 223> Locus 1-ST-4 <400> 36 tgttgcgact acgtgaataa t 21 <210> 37 <211> 74 <212> DNA <213> Artificial <220> <223> Locus 2-ST <400> 37 gtacgacaac taccccatac caaaccttcc ttcgtacccc tatagtgagt cgtattattt 60 gccagccatt ctag 74 <210> 38 <211> 21 <212> DNA <213> Artificial <220> <223> Locus 2-ST-4 <400> 38 cagcatcacc gccataataa t 21 <210> 39 <211> 74 <212> DNA <213> Artificial <220> <223> Locus 3-ST <400> 39 gtacgacaac taccccatac caaaccttcc ttcgtacccc tatagtgagt cgtattattg 60 ctttagtggc agta 74 <210> 40 <211> 19 <212> DNA <213> Artificial <22 0 > <223> Locus 3-ST-4 <400> 40 tgtgttacat tgtattaat 19 <210> 41 <211> 74 <212> DNA <213> Artificial <220> <223> Locus 4-ST <400> 41 gtacgacaac taccccatac caaaccttcc ttcgtacccc tatagtgagt cgtattattt 60 gttacattgt atgc 74 <210> 42 <211> 21 <212> DNA <213> Artificial <220> <223> Locus 4-ST-4 <400> 42 tctgccgaaa gcttgaataa t 21 <210> 43 <211> 74 <212> DNA <213> Artificial <220> <223> Locus 5-ST <400> 43 gtacgacaac taccccatac caaaccttcc ttcgtacccc tatagtgagt cgtattatta 60 gttcctggtc ccca 74 <210> 44 <211> 21 <212> DNA <213> < 220> <223> Locus 5-ST-4 <400> 44 gttccttgtc tgattaataa t 21 <210> 45 <211> 75 <212> DNA <213> Artificial <220> <223> Signal template-G <400> 45 ggatccattc gttacctggc tctcgccagt cgggatcccc ctatagtgag tcgtattatt 60 agttgacacc ctgat 75 <210> 46 <211 > 75 <212> DNA <213> Artificial <220> <223> Signal template-A <400> 46 ggatccattc gttacctggc tctcgccagt cgggatcccc ctatagtgag tcgtattatt 60 agttaacacc ctgat 75 <210> 47 <211> 75 <212> DNA <213> ial <220> <223> Signal template-T <400> 47 ggatccattc gttacctggc tctcgccagt cgggatcccc ctatagtgag tcgtattatt 60 agtttacacc ctgat 75 <210> 48 <211> 75 <212> DNA <213> Artificial <220> <223> Signal template-C <400> 48 ggatccattc gttacctggc tctcgccagt cgggatcccc ctatagtgag tcgtattatt 60 agttcacacc ctgat 75 <210> 49 <211> 21 <212> DNA <213> Artificial <220> <223> D614G Mutation ST-4 <400> 49 caggg acttc tgtgcaataa t 21 < 210> 50 <211> 74 <212> DNA <213> Artificial <220> <223> E. coli L1-ST <400> 50 gtacgacaac taccccatac caaaccttcc ttcgtacccc tatagtgagt cgtattattc 60 gggtaacgtc aatg 74 <210> 51 <211> 21 < 212> DNA <213> Artificial <220> <223> E. coli L1-ST-4 <400> 51 ccggtgcttc ttctgaataa t 21 <210> 52 <211> 74 <212> DNA <213> Artificial <220> <223 > E. coli L2-ST <400> 52 gtacgacaac tacccatac caaaccttcc ttcgtacccc tatagtgagt cgtattattc 60 cacgctttcg cacc 74 <210> 53 <211> 21 <212> DNA <213> Artificial <220> <223> E. coli L2-ST- 4 <400> 53 aatcctgttt gctccaataa t 21 <210> 54 <211> 74 <212> DNA <213> Artificial <220> <223> S. aureus L1-ST <400> 54 gtacgacaac taccccatac caaaccttcc ttcgtacccc tatagtgagt cgtattattc 6 0 gctttcgcac atca 74 <210> 55 <211> 21 <212> DNA <213> Artificial <220> <223> S. aureus L1-ST-4 <400> 55 cctgtttgat ccccaaataa t 21 <210> 56 <211> 74 <212> DNA <213> Artificial <220> <223> S. aureus L2-ST <400> 56 gtacgacaac taccccatac caaaccttcc ttcgtacccc tatagtgagt cgtattattg 60 gacttaaccc aaca 74 <210> 57 <211> 21 <212> DNA <213> Artificial <220> <223> S. aureus L2-ST-4 <400> 57 gttgcgctcg ttgcgaataa t 21 <210> 58 <211> 40 <212> DNA <213> Artificial <220> <223> SARS-CoV-2 N gene_IVT-FP <400> 58 taatacgact cactataggg gcagtcaagc ctcttctcgt 40 <210> 59 <211> 20 <212> DNA <213> Artificial <220> <223> SARS-CoV-2 N gene_IVT-RP <400> 59 aacattggcc gcaaattgca 20 <210> 60 <211> 40 <212> DNA <213> Artificial <220> <223> SARS-CoV-1_IVT-FP <400> 60 taatacgact cactataggg gcagtcaagc ctcttctcgc 40 <210> 61 <211> 20 <212> DNA <213> Artificial <220> <223> SARS-CoV-1_IVT-RP <400> 61 ggtgtgactt ccatgccaat 20 <210> 62 <211> 40 <212> DNA <213> Artificial <220> <223> MERS-CoV_IVT-FP <400 > 62 taatacgact cactataggg atagtcaatc atcttcaaga 40 <210> 63 <211> 20 <212> DNA <213> Artificial <220> <223> MERS-CoV_IVT-RP <400> 63 ttctgatggg taagtttaaa 20 <210> 64 <211> 40 < 212> DNA <213> Artificial <220> <223> HCoV-229E-FP <400> 64 taatacgact cactataggg gtgctccttc ccggtctcag 40 <210> 65 <211> 20 <212> DNA <213> Artificial <220> <223> HCoV -229E-RP <400> 65 cttccaaagt tgtggtcaag 20 <210> 66 <211> 40 <212> DNA <213> Artificial <220> <223> HCoV-NL63-FP <400> 66 taatacgact cactataggg gctctaataa ctcatctcgt 40 <210> 67 <211> 20 <212> DNA <213> Artificial <220> <223> HCoV-NL63-RP <400> 67 tcccccatat tgtgattaaa 20 <210> 68 <211> 40 <212> DNA <213> Artificial <220> <223> HCoV-OC43-FP <400> 68 taatacgact cactataggg ctgctcctaa ttccagatct 40 <210> 69 <211> 20 <212> DNA <213> Artificial <220> <223> HCoV-OC43-RP <400> 69 aactctaatc ttgatccaaa 20 <210> 70 <211> 40 <212> DNA <213> Artificial <220> <223> HCoV-HKU1-FP <400> 70 taatacgact cactataggg ctgcttctaa tagtcgacca 40 <210> 71 <211> 20 <212> DNA < 213> Artificial <220> <223> HCoV-HKU1-RP <400> 71 aagtctaatt tagaaccaaa 20 <210> 72 <211> 70 <212> DNA <213> Artificial <220> <223> SARS-CoV-2 S gene (G614)_IVT-FP <400> 72 taatacgact cactataggg tctaaccagg ttgctgttct ttatcagggt gttaactgca 60 cagaagtccc 70 <210> 73 <211> 50 <212> DNA <213> Artificial <220> <223> SARS-CoV-2 S gene (G614 )_IVT-RP <400> 73 ggagtaagtt gatctgcatg aatagcaaca gggacttctg tgcagttaac 50 <210> 74 <211> 70 <212> DNA <213> Artificial <220> <223> SARS-CoV-2 S gene (D614)_IVT-FP < 400> 74 taatacgact cactataggg tctaaccagg ttgctgttct ttatcaggat gttaactgca 60 cagaagtccc 70 <210> 75 <211> 50 <212> DNA <213> Artificial <220> <223> SARS-CoV-2 S gene (D614)_IVT-RP <400> 75 ggagtaagtt gatctgcatg aatagcaaca gggacttctg tgcagttaac 50 <210> 76 <211> 380 <212> RNA <213> Artificial <220> <223> SARS-CoV-2 N gene <400> 76 gcagucaagc cucuucucgu uccucaucac guagucgcaa caguucaaga a auucacacuc 60 caggcagcag uaggggaacu ucuccugcua gaauggcugg caauggcggu gaugcugcuc 120 uugcuuugcu gcugcuugac agauugaacc agcuugagag caaaaugucu gguaaaggcc 180 aacaacaaca aggccaaacu gucacuaaga aaucugcugc ugaggcuucu aagaagccuc 240 ggcaaaaacg uacugccacu aaagcauaca ca agcuuucggc agacgugguc 300 cagaacaaac ccaaggaaau uuuggggacc aggaacuaau cagacaagga acugauuaca 360 aacauuggcc gcaaauugca 380 <210> 77 <211> 442 <212> RNA <213> Artificial <220> <223> SARS-CoV-1 <400> 77 gcagucaagc cucuucucgc uccucaucac guagucgcgg uaauucaaga aauucaacuc 60 cuggcagcag uaggggaaau ucuccugcuc gaauggcuag cggagguggu gaaacugccc 120 ucgcgcuauu gcugcuagac ag auugaacc agcuugagag caaaguuucu gguaaaggcc 180 aacaacaaca aggccaaacu gucacuaaga aaucugcugc ugaggcaucu aaaaagccuc 240 gccaaaaacg uacugccaca aaacaguaca acgucacuca agcauuuggg agacgugguc 300 cagaacaaac ccaaggaaau uucggggacc aagaccuaau cagacaagga acugauuaca 360 aacauuggcc gcaaauugca caauuugcuc caagugccuc ugcauucuuu ggaaugucac 420 gcauggcau ggaagucaca cc 442 <210> 78 <211> 451 <212> RNA > Artificial <220> <223> MERS-CoV <400> 78 auagucaauc aucuucaaga gccucuagcu uaagcagaaa cucuuccaga ucuaguucac 60 aagguucaag aucaggaaac ucuacccgcg gcacuucucc agguccaucu ggaaucggag 120 caguaggagg ugaucuacuu uaccuugauc uucugaacag acuacaagcc cuugagucug 180 gcaaaguaaa gcaaucgcag ccaaaaguaa ucac uaagaa agaugcugcu gcugcuaaaa 240 auaagaugcg ccacaagcgc acuuccacca aaaguuucaa cauggugcaa gcuuuugguc 300 uucgcggacc aggagaccuc cagggaaacu uuggugaucu ucaauugaau aaacucggca 360 cugaggaccc acguuggccc caaauugcug agcuugccagu gcuuuuaugg 420 guaugucgca auuuaaacuu acccaucaga a 451 <210> 79 <211> 391 <212> RNA <213> Artificial <220> <223> HCoV-229E <400> 79 gugcuccuuc ccggucucag ucgaggucgc agagucgcgg ucguggugaa uccaaaccuc 60 aaucucggaa uccuucaagu gacagaaacc auaaca guca ggaugacauc augaaggcag 120 uugcugcggc ucuuaaaucu uuagguuuug acaagccuca ggaaaaagau aaaaagucag 180 cgaaaacggg uacuccuaag ccuucucgua aucagagucc ugcuucuucu caaacuucug 240 ccaagagucu ugcucguucu cagaguucug aaacaaaaga acaaaagcau gaaaugcaaa 300 agccacggug gaaaagacag ccuaaugaug augugacauc uaugucaca caauguuuug 360 gccccagaga ccuugaccac aacuuuggaa g 391 <210> 80 <211> 355 <212> RNA <213> Artificial <220> <223> HCoV-NL63 <400> 80 gcucuaauaa cucaucucgu gcuagcaguc guucuucaac ucguaacaac ucacgagacu 60 cuucucguag cacuucaaga caacagucuc gcacucguuc ugaauucuaac cagucuucuu 120 cagaucuugu ugcugcuguu acuuuggcuu uaaagaacuu agguuuugau a accagucga 180 agucaccuag uucuucuggu acuuccacuc cuaagaaacc uaauaagccu cuuucucaac 240 ccagggcuga uaagccuucu caguugaaga aaccucguug gaagcguguu ccuaccagag 300 aggaaaaugu uauucagugc uuugguccuc gugauuuuaa ucacaauaug gggga 355 < 210> 81 <211> 412 <212> RNA <213> Artificial <220> <223> HCoV-OC43 <400> 81 cugcuccuaa uuccagaucu acuucgcgca cauccagcag agccucuagu gcaggaucgc 60 guaguagagc caauucuggc aauagaaccc cuaccucugg uguaacaccu gacauggcug 120 aucaaauugc uagucuuguu cuggcaaaac uuggcaagga ugccacuaaa ccucagcaag 180 uaacuaagca uacugccaaa gaagucagac agaaaauuuu gaauaagccc cgccagaaga 240 ggagccccaa uaaaacaaugc acuguucagc aguguuuugg uaagagaggc ccuaaucaga 300 auuuuggugg uggagaaaug uuaaaacuug gaacuaguga cccacaguuc cccauucuug 360 cagaacucgc accca cagcu ggugcguuuu ucuuuggauc aagauuagag uu 412 <210> 82 <211> 409 <212> RNA <213> Artificial <220> <223> HCoV -HKU1 <400> 82 cugcuucuaa uagucgacca gguucacguu cucaaucacg uggacccaau aaucguucau 60 uaaguagaag uaauucuaau uuuagacauu cagauucuau aguaaaaccu gauauggcug 120 augagaucgc uaaucuuguu uuagccaagc uugguaaaga uucuaaaccu cagcaa guca 180 cuaagcaaaa ugccaaggaa aucaggcaua aaauuuuaac aaaaccucgc caaaagcgaa 240 cuccuaauaa acauuguaau guucaacagu guuuugguaa aagaggaccu ucucaaaauu 300 uugguaaugc ugaaauguua aagcuuggua cuaaugaucc ucaguuuccu auucuugcag 360 aauuagcucc uacaccaggu gcuuuuuucu uugguucuaa auuagacuu 409 <210> 83 <211> 80 <212> RNA <213> Artificial <220> <223> SARS-CoV-2 S gene (G614) <400> 83 ucuaaccagg uugcuguucu uuaucagggu guuaacugca cagaaguccc uguugcuauu 60 ca ugcagauc aacuuacucc 80 <210> 84 <211> 80 <212> RNA <213> Artificial <220> <223> SARS-CoV-2 S gene (D614) <400> 84 ucuaaccagg uugcuguucu uuaucaggau guuaacugca cagaaguccc uguugcuauu 60 caugcagauc aacuuacucc 80 <210> 85 <211> 610 <212> RNA <213> Artificial <220> <223> E. coli 16S rRNA <220> <221> misc_feature <222> (66)..(66) <223> n is a, c, G, or U <220> <221> MISC_FEATURE <222> (526) .. (527) <223> n is a, C, G, or U <400> 60 CAUGCGU Guaugaaa ggcuucgg uuguaagua cuuucagcgg ggaggaaggg 120 aguaaaguua auaccuuugc ucauugacgu uacccgcaga agaagcaccg gcuaacuccg 180 ugccagcagc cgcgguaaua cggagggugc aagcguuau cggaauuacu gggcguaaag 240 cgcacgcagg cgguuuguua agucagaugu gaaauccccg ggcucaaccu gggaacugca 300 ucugauacug gcaagcuuga gucucguaga gggggguaga auuccaggug uagcggugaa 360 augcguagag aucuggagga auaccggugg cgaaggcggc ccccuggacg aagacugacg 420 cucaggugcg aaagcguggg gagcaaacag gauuagauac ccugguaguc ccguaa 480 acgaugucga cuuggagguu gugcccuuga ggcguggcuu ccggannuaa cgcguuaagu 540 cgaccgccug gggaguacgg ccgcaagguu aaaacucaaa ugaauugacg ggggccgcac 600 aagcggugga 610 <210> 86 <211> 524 <212> RNA <213> Artificial <220> <223> S. aureus 16S rRNA <400> cagaagagga aaguggaauu ccauguguag cggugaaaug cgcagagaua uggaggaaca 60 ccaguggcga aggcgacuuu cuggucugua acugacgcug augugcgaaa gcguggggau 120 caaacaggau uagauacccu gguaguccac gccguaaacg augagugcua aguguuaggg 180 gguuucccgc cccuuagugc ugcagcuaac gcauuaagca cuccgccugg ggaguacgac 240 cgcaagguug aaag gaauugacgg ggacccgcac aagcggugga gcaugugguu 300 uaauucgaag caacgcgaag aaccuuacca aaucuugaca uccuuugaca acucuagaga 360 uagagccuuc cccuucgggg gacaaaguga cagguggugc augguugucg ucagcucgug 420 ucgugagaug uguuaag ucccgca acg agcgcaaccc uuaagcuuag uugccaucau 480uaaguugggc acucuaaguu gacugccggu gacaaaccgg agga 524

Claims (23)

An isothermal one-pot reaction probe set for detecting one or more target molecules, including a first probe and a second probe,
The first probe is a promoter probe (PP) having a structure comprising the following general formulas I and II,
[General Formula I]
5'-XYZ-3'
[General formula II]
3'-Ya-5'
In the above general formula I,
X is a region of the aptamer sequence with an interactive labeling system comprising one label or multiple labels that generates a detectable signal;
Y is a sequence complementary to the T7 promoter;
Z is a site that binds or connects to the target molecule;
X and Y are deoxyribonucleotides;
In the general formula II,
Ya is a partial sequence of the T7 promoter; Ya is a deoxyribonucleotide;
The second probe is a promoter probe (PP) having a structure of the following general formula (III),
[General formula III]
3'-Yb-Z'-5'
In the general formula (III),
Yb is a partial sequence of the T7 promoter and is a deoxyribonucleotide;
Z' is a site that binds or connects to the target molecule;
The Ya:Yb split ratio is 19:1 to 16:4,
The first probe and the second probe are characterized in that transcription is initiated by a polymerase after binding or linking to the target molecule to generate a signal,
Isothermal single reaction probe set for target molecule detection.
According to paragraph 1,
An isothermal single reaction probe set for detecting target molecules, wherein the target molecule is at least one selected from the group consisting of nucleic acids, proteins, cells, and ATP.
According to paragraph 2,
When the target molecule is a nucleic acid, Z and Z' are nucleic acid sequences that bind complementary to the nucleic acid to be detected,
When the target molecule is a protein, Z and Z' are antibodies or aptamers that can bind to the protein,
When the target molecule is a cell, Z and Z' are an antibody or aptamer that can bind to one or more types selected from the group consisting of extracellular proteins, cellular phospholipids, bacterial peptidoglycan, and LPS,
When the target molecule is ATP, Z and Z' are split aptamers capable of binding to ATP,
Isothermal single reaction probe set for target molecule detection.
According to paragraph 1,
The Ya and Yb are partial sequences of the T7 promoter,
When Ya and Yb are sequentially arranged in the order of Ya and Yb, they form the T7 promoter,
An isothermal single reaction probe set for detecting target molecules, characterized in that the Ya:Yb split ratio is 19:1 to 16:4.
According to paragraph 4,
An isothermal single reaction probe set for detecting target molecules, characterized in that the Ya:Yb split ratio is 16:4.
According to paragraph 1,
An isothermal single reaction probe set for detecting a target molecule, wherein the label is selected from the group consisting of a chemical label, an enzyme label, a radioactive label, a fluorescent label, a luminescent label, a chemiluminescent label, and a metal label.
According to paragraph 1,
The general formula I of the first probe may be modified into general formula I', which additionally includes an overlapping sequence W between Y and Z;
[General formula I']
5'-XYWZ-3'
The general formula III of the second probe can be transformed into general formula III', which additionally includes an overlapping sequence W' between Yb and Z';
[Formula III']
3'-Yb-W'-Z'-5'
Isothermal single reaction probe set for target molecule detection.
In clause 7,
An isothermal single reaction probe set for detecting target molecules, characterized in that the length of the overlapping sequences W and W' is 1bp or 2bp.
According to paragraph 1,
An isothermal single reaction probe set for detecting target molecules, characterized in that the isothermal single reaction is performed at a constant temperature in the range of 15°C to 50°C.
According to paragraph 1,
The probe set is an isothermal single reaction probe set for detecting target molecules, characterized in that it detects circular RNA separately from linear RNA.
According to paragraph 1,
The polymerase is bacteriophage T7 RNA polymerase, bacteriophage T3 polymerase, bacteriophage RNA polymerase, bacteriophage ΦII polymerase, Salmonella bacteriophage sp6 polymerase, Pseudomonas bacteriophage gh-1 polymerase, E. coli RNA polymerase holoenzyme. (holoenzyme), E. coli RNA polymerase core enzyme, human RNA polymerase I, human RNA polymerase II, human RNA polymerase III, human mitochondrial RNA polymerase and variants thereof. An isothermal single reaction probe set for detecting target molecules.
According to paragraph 1,
The isothermal single reaction is characterized in that it is carried out simultaneously in a single reaction solution containing Tris-HCl, MgCl ₂ , DTT, spermidine, rNTPs, RNase inhibitor, and SSB (Single-Stranded DNA Binding Protein). Isothermal single reaction probe set for molecular detection.
A composition for detecting a target molecule, comprising an isothermal single reaction probe set for detecting a target molecule including the first probe and the second probe of claim 1.
According to clause 13,
The composition is for detecting two or more target molecules, characterized in that it includes an isothermal single reaction probe set for detecting two or more target molecules.
According to clause 14,
The isothermal single reaction probe sets for detecting two or more target molecules each include different interactive labeling systems; Each of the two or more probe sets binds to a different target molecule; A composition for detecting target molecules, which allows multiple detection of different target molecules.
According to clause 13,
A composition for detecting a target molecule, wherein the isothermal single reaction is performed at a constant temperature in the range of 15°C to 50°C.
A kit for detecting target molecules, comprising the composition for detecting target molecules according to any one of claims 13 to 16, a polymerase, and an isothermal single reaction solution.
A method of detecting a target molecule using the isothermal single reaction probe set for detecting the target molecule of any one of claims 1 to 12.
According to clause 18,
A method of detecting a target molecule comprising performing an isothermal nucleic acid amplification reaction prior to the method of detecting the target molecule.
According to clause 19,
A method for detecting a target molecule, wherein the isothermal nucleic acid amplification reaction is RPA (Recombinase Polymerase Amplification).
An on-site molecular diagnostic method using the isothermal single reaction probe set for detecting target molecules according to any one of claims 1 to 12.
According to clause 21,
The molecular diagnostic method is an on-site molecular diagnostic method, characterized in that it is used to detect pathogenic microorganisms.
According to clause 22,
The pathogenic microorganisms include Staphylococcus Aureus, Vibrio vulnificus, E. coli, Middle East Respiratory Syndrome Coronavirus, and Influenza A virus. virus), Severe Acute Respiratory Syndrome Coronavirus, Respiratory Syncytial Virus (RSV), Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV), Human Papilloma Virus (HPV), Human Parainfluenza Viruses (HPIV), dengue virus, Hepatitis B Virus (HBV), yellow fever virus, rabies virus, Plasmodium, cytomegalovirus (CMV), Mycobacterium tuberculosis, Chlamydia trachomatis, Rota Viruses, human metapneumovirus (hMPV), Crimean-Congo hemorrhagic fever virus, Ebola virus, Zika virus, henipavirus, norovirus, Lassa virus, rhinovirus, flavivirus, Rift Valley fever Viruses, hand, foot and mouth disease virus, Salmonella sp., Shigella sp., Enterobacteriaceae sp., Pseudomonas sp., Moraxella sp., Helicobacter sp. ) and Stenotrophomonas sp., an on-site molecular diagnostic method, characterized in that one or more species selected from the group consisting of.