KR20250040124A - Method of detecting food poisoning Bacteria using probe for primer generation rolling circle amplification - Google Patents

Abstract

The present invention relates to a method for detecting food poisoning bacteria, a composition for detecting food poisoning bacteria, and a kit for detecting food poisoning bacteria.
According to the present invention, by optimizing the primer formation rotational amplification reaction, the speed and accuracy of detection of food poisoning bacteria can be increased, and thus, it can be usefully utilized for detection of food poisoning bacteria in various fields such as agricultural and marine products and food.

Description

{Method of detecting food poisoning bacteria using probe for primer generation rolling circle amplification}

The present invention relates to a method for detecting food poisoning bacteria, a composition for detecting food poisoning bacteria, and a kit for detecting food poisoning bacteria.

DNA amplification is a key method that has contributed to the rapid development of biotechnology and molecular biology. PCR (Polymerase Chain Reaction), the most basic and oldest method of DNA amplification, has been used for various purposes in the fields of medicine, agriculture, environment, and food, such as gene cloning, disease diagnosis, and bacteria or virus detection. Real-time PCR (RT-PCR) is a quantitative analysis method that can monitor the fluorescence signal of the amplicon generated during the PCR process in real time. RT-PCR has expanded the capabilities of PCR and has been widely applied to the analysis of various biological samples due to its excellent sensitivity and specificity, and has recently served as a common tool used as a global standard for SARS-CoV-2 testing.

However, RT-PCR requires expensive equipment with access to thermal cycling and fluorescence measurements, and existing standardized protocols and kits for RT-PCR are not available for all types of targets, so high expertise and technology are required to develop new qPCR assays for new targets.

With the advancement of DNA amplification research, loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinant enzyme polymerase amplification (RPA), and strand displacement amplification (SDA) have been developed, and these methods are isothermal amplification methods that do not require a thermal cycler. Although the principles of these methods are relatively complex compared to PCR, they often provide better sensitivity and accuracy than PCR, and have potential applications in terms of speed, cost, scale, versatility, or portability. Therefore, isothermal amplification methods have recently been expanded to various biosensors as an attractive and effective detection strategy, and their applicability has been proven. RCA, one of the isothermal DNA amplification methods, uses a circular single-stranded DNA probe as a template for DNA polymerization, and enables amplification of the template DNA sequence in the presence of a primer at a relatively low (30°C) single temperature. The simplicity and efficiency of RCA technology have enabled the development of various detection methods to be expanded to field analysis, and it is being applied to the analysis of various target substances such as not only chemicals but also biomolecules.

The RCA-based detection method for detecting target DNA uses a linear padlock probe, which is hybridized with the target DNA and then ligated by T4 DNA ligase to form a circular probe that can be used as a template in the subsequent RCA. After the ligation reaction, the RCA reaction by DNA polymerization occurs at the 3' end of the target DNA or an additionally added primer. By including a ligation process before RCA, padlock probes that hybridize only to the sequence of the target DNA are selectively circularized. However, when both the ligation and RCA steps are included, the padlock probe linked to a long linear single-stranded DNA can inhibit RCA, which may result in limited applications in detecting short artificially synthesized DNA or single nucleotide polymorphisms.

Against this backdrop, the inventors of the present invention studied an amplification method capable of detecting food poisoning bacteria more simply and accurately, and completed the present invention by confirming the detection of food poisoning bacteria by the PG-RCA method that omits the ligation step and can simply convert linear RCA into exponential mode.

Chinese Publication Patent No. 113755557

An exemplary object of the present invention is to provide a method for detecting food poisoning bacteria comprising the following steps.

a) A step of sonicating the genomic DNA of food poisoning bacteria;

b) a step of hybridizing the sonicated genomic DNA and the circular probe pair;

c) a step of amplifying the target nucleic acid by mixing dNTP and DNA polymerase into the hybridized reaction product; and

d) A step of detecting the target nucleic acid.

Another exemplary object of the present invention is to provide a composition for detecting food poisoning bacteria, comprising a pair of circular probes including a nucleic acid sequence complementary to the genomic DNA of the food poisoning bacteria.

Another exemplary object of the present invention is to provide a kit for detecting food poisoning bacteria, comprising the composition.

The technical problems to be achieved according to the technical idea of the invention disclosed in this specification are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

This is explained specifically as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of this application cannot be considered limited by the specific description described below.

As one aspect for achieving the above object, the present invention provides a method for detecting food poisoning bacteria comprising the following steps.

a) A step of sonicating the genomic DNA of food poisoning bacteria;

b) a step of hybridizing the sonicated genomic DNA and the circular probe pair;

c) a step of amplifying the target nucleic acid by mixing dNTP and DNA polymerase into the hybridized reaction product; and

d) A step of detecting the target nucleic acid.

The term "circular probe" of the present invention refers to a probe manufactured by connecting the 3' and 5' ends of a linear single-stranded DNA containing a base sequence complementary to a target nucleic acid, and includes a hybridization sequence for the target nucleic acid and a complementary sequence of a nicking site.

In the present invention, amplification of the target nucleic acid may be performed by primer generation rolling circle amplification (PG-RCA).

The term "primer-forming rolling circle amplification" of the present invention is an improvement on rolling circle amplification, which is a nucleic acid amplification reaction that amplifies a circular nucleic acid template through a rolling circle mechanism, and includes a nicking endoneuclease site in a circular probe to continuously form primers during the amplification process, thereby enhancing amplification of target nucleic acids.

In one embodiment, each probe (Forward (F) and Reverse (R)) in the circular probe pair binds to the sense or antisense site of the target nucleic acid and hybridizes with the target nucleic acid. DNA polymerase then cleaves the tail that is pushed out from the 3' end of the target nucleic acid, and a rolling-circle amplification reaction is initiated at the 3' end of the target nucleic acid, and a linked sequence copy of the circular probe binding site and the nicking site is amplified to generate primers required for additional rolling-circle amplification. In this manner, multiple reaction cycles are performed simultaneously, resulting in an exponential accumulation of primers and reaction products over time.

Specifically, in an embodiment of the present invention, it was confirmed that the amount of amplification product increased more when a pair of circular probes was used compared to a single circular probe when detecting E. coli.

In the present invention, the circular probe may include a binding site complementary to a base sequence of 10 to 30 bp in the target nucleic acid, and specifically, may include a binding site complementary to a base sequence of 15 to 25 bp.

In the present invention, the circular probe may include a nicking endonuclease site within the probe. The nicking endonuclease site is cleaved by a nicking enzyme to form a new primer.

In the present invention, the concentration of the circular probe may be 1 μM to 2 μM, specifically 1 μM to 1.5 μM or 1.5 μM to 2 μM, but is not limited thereto.

In the present invention, the concentration of the dNTP may be 0.05 mM to 0.2 mM, specifically 0.05 mM to 0.15 mM, and more specifically 0.05 mM to 0.1 mM, 0.1 mM to 0.2 mM or 0.1 mM to 0.15 mM, but is not limited thereto.

In the present invention, the concentration of the DNA polymerase may be 2 U/ul to 10 U/ul, specifically 4 U/ul to 8 U/ul, and more specifically 5 U/ul to 7 U/ul, but is not limited thereto.

In one embodiment of the present invention, the DNA polymerase may be phi29 DNA polymerase, but is not limited thereto, and any enzyme capable of performing rotational circle amplification using the nucleic acid sequence of the probe as a template may be appropriately selected and used by those skilled in the art.

In the present invention, the food poisoning bacteria may be at least one selected from the group consisting of Escherichia coli ( e. coli ), Salmonella Sp . , Bacillus Sp., Campylobacter Sp., Staphylococcus Sp., and Listeria Sp.

In one embodiment of the present invention, when the food poisoning bacteria is E. coli, the circular probe pair may be a probe pair represented by sequence numbers 1 and 2.

In one embodiment of the present invention, when the food poisoning bacteria is Salmonella genus, the circular probe pair may be a probe pair represented by sequence numbers 3 and 4.

In one embodiment of the present invention, when the food poisoning bacteria is of the genus Bacillus, the circular probe pair may be a probe pair represented by sequence numbers 5 and 6.

The term "target nucleic acid" of the present invention means all types of nucleic acids to be detected, and may be characterized by, but is not 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, and may be a mutant base sequence including a variation in the base sequence, and the mutation may be characterized by, but is not limited to, being selected from the group consisting of single nucleotide polymorphism (SNP), insertion, deletion, point mutation, fusion mutation, translocation, inversion, and LOH (loss of heterozygosity).

The term "nucleic acid" of the present invention refers to a nucleotide polymer, and unless otherwise limited, includes known analogs of natural nucleotides that can function in a similar manner (e.g., hybridizing) to naturally occurring nucleotides. The term nucleic acid includes, for example, genomic DNA; complementary DNA (cDNA) (which is the DNA representation of mRNA, usually obtained by reverse transcription or amplification of messenger RNA (mRNA)); DNA molecules produced synthetically or by amplification; and any form of DNA or RNA, including mRNA, and includes single-stranded molecules as well as double- or triple-stranded nucleic acids. The nucleic acid strands in a double- or triple-stranded nucleic acid 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 "hybridization" of the present invention means the formation of a double-stranded nucleic acid by hydrogen bonding between single-stranded nucleic acids having complementary base sequences, and is used in a similar meaning to annealing. However, in a slightly broader sense, hybridization includes not only the case where the base sequences between two single-strands are completely complementary (perfect match), but also, exceptionally, the case where some base sequences are not complementary (mismatch).

The term "complementary" of the present invention refers to the ability for exact pairing between two nucleotides. That is, if a nucleotide at a given position in a nucleic acid can hydrogen bond with a nucleotide in another nucleic acid, then 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 it may be complete, where full complementarity exists between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between the nucleic acid strands.

As another aspect for achieving the above object, the present invention provides a composition for detecting food poisoning bacteria, comprising a pair of circular probes including a nucleic acid sequence complementary to a food poisoning bacteria.

The terms circular probe and food poisoning bacteria of the present invention are as described above.

In the present invention, when the food poisoning bacteria is Escherichia coli, the circular probe pair may be a probe pair represented by sequence numbers 1 and 2, when the food poisoning bacteria is Salmonella genus, the circular probe pair may be a probe pair represented by sequence numbers 3 and 4, and when the food poisoning bacteria is Bacillus genus, the circular probe pair may be a probe pair represented by sequence numbers 5 and 6.

In the present invention, the composition may contain dNTP and DNA polymerase.

As another aspect for achieving the above purpose, the present invention provides a kit for detecting food poisoning bacteria comprising the composition.

In the present invention, the kit may be for primer forming rotational amplification.

In one embodiment of the present invention, the kit may include, but is not limited to, reagents provided in separate glass containers, plastic containers, or strips of plastic or paper.

In the present invention, the kit may additionally include materials typically required for primer formation rolling-circle amplification reaction and base sequence analysis. For example, it may include a primer formation rolling-circle amplification reaction mixture including at least one selected from the group consisting of dNTP, DNA polymerase, ligase, reaction buffer, distilled water, etc. In addition, the kit may be manufactured into a plurality of separate packagings or compartments including the above-described reagent components.

According to the present invention, by optimizing the primer formation rotational amplification reaction, the speed and accuracy of detection of food poisoning bacteria can be increased, and thus, it can be usefully utilized for detection of food poisoning bacteria in various fields such as agricultural and marine products and food.

Figure 1 is an overall schematic diagram of a method for detecting a target gene using primer-forming rolling circle amplification.
Figure 2 shows the results of amplifying each of the VT2, ttr, and hblD genes and confirming them by gel electrophoresis.
Figure 3 shows the results of manufacturing a circular probe of the present invention and confirming it through gel electrophoresis.
Figure 4 shows the results of detecting E. coli O157:H7 using a single probe or a probe pair in the linear RCA or PG-RCA method.
Figure 5 shows the results of amplification by PG-RCA by varying the concentration of DNA polymerase under 1 mM dNTP conditions in the presence or absence of genomic DNA.
Figure 6 shows the results of confirming amplification by PG-RCA by varying the concentration of DNA polymerase under 0.1 mM dNTP conditions in the presence or absence of genomic DNA.
Figure 7 shows the results of confirming amplification by PG-RCA by changing the concentration of the circular probe (1.5, 0.5, 0.25, and 0.125 μM).
Figure 8 shows the results of comparing the amplification by PG-RCA of a control probe containing a non-specific sequence for food poisoning bacteria and probes having sequences specific for Escherichia coli O157:H7, Salmonella typhimurium, and Bacillus cereus, respectively.
Figure 9 shows the PG-RCA results according to the concentration of food poisoning bacteria (E. coli O157:H7, Salmonella Typhimurium, Bacillus cereus) genomic DNA, expressed as fluorescence intensity.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are intended to exemplify the present invention, and the scope of the present invention is not limited to these examples.

Example 1: Manufacturing of circular probes

1-1. Preparation of compounds and reagents

DNA probes were manufactured by order of Bioneer (Daejeon, Korea). Phi29 DNA polymerase (10 U/μL), 10 mM dNTP, 5X T4 DNA ligase buffer, sodium dodecyl sulfate (SDS) solution (10% [w/v]), and polyacrylamide gel (15%) were purchased from Thermo Fisher Scientific (Massachusetts, USA). 10x RCA buffer contains 330 mM Tris-acetate (pH 7.9), 100 mM Mg-acetate, 660 mM K-acetate, 1% (v/v) Tween 20, and 10 mM DTT. Exonuclease I, Exonuclease III, and NB.Bbvcl were purchased from New England Biolabs (OR, USA). The ssDNA Ligation kit containing 1 mM ATP, 50 mM MnCl ₂ , 10X Ligase Buffer, and 100 U/μL CircLigase ssDNA Ligase was purchased from Biosearch Technologies (Hoddesdon, UK).

SYBR Green I & II and TBE-Urea gels (15%, 12-well) were purchased from Invitrogen (MA, USA). TBE/Urea Sample buffer was purchased from Bio-Rad (CA, USA). 20-bp DNA ladder was purchased from Takara (Kyoto, Japan). Oligo Clean-Up and Concentration Kit was purchased from Norgen Biotek (Canada).

1-2. Probe Manufacturing

Figure 1 is an overall schematic diagram of a target gene detection method using primer-forming rolling circle amplification. VT2, ttr, and hblD genes were selected as target genes for detecting E. coli O157:H7 (E), Salmonella typhimurium (S), and Bacillus cereus (B), respectively. Linear single-stranded DNA (ssDNA) was prepared so that it contained 20 to 23 bp of complementary base sequences to each target gene and had a Tm value of 57.2 to 57.8°C so that it could be annealed simultaneously at the same temperature for three target genes (Table 1).

Target
Food poisoning bacteria Oligo name Sequence (5'-3') Sequence number E. coli O157:H7 Cir_VT2 F (Phosphate)- GAAACTGTCC CCTCAGC TATATGCACATGAAACCCATCCCGCCCAACCC CCTCAGC AAACCCATCCCGCCCAACCC GGCACTGTCT 1 Cir_VT2 R (Phosphate)- CTGACATTCTG CCTCAGC TATATGCACATGAAACCCATCCCGCCCAACCC CCTCAGC AAACCCATCCCGCCCAACCC TCGCCAGTTAT 2 Salmonella Typhimurium Cir_ttr F (Phosphate)- ATTACAACATGG CCTCAGC TATATGCACATGAAACCCATCCCGCCCAACCC CCTCAGC AAACCCATCCCGCCCAACCC CTCACCAGGAG 3 Cir_ttr R (Phosphate)- AAGTGACCAT CCTCAGC TATATGCACATGAAACCCATCCCGCCCAACCC CCTCAGC AAACCCATCCCGCCCAACCC GCTCAGACCAA 4 Bacillus cereus Cir_hblD F (Phosphate)- TATTCATGCATT CCTCAGC TATATGCACATGAAACCCATCCCGCCCAACCC CCTCAGC AAACCCATCCCGCCCAACCC ACCGGTAACAC 5 Cir_hblD R (Phosphate)- GCTTAGATGCTG CCTCAGC TATATGCACATGAAACCCATCCCGCCCAACCC CCTCAGC AAACCCATCCCGCCCAACCC GGAGTCCATAT 6 Cir_control (Phosphate)- TTTTTTGGGGG CCTCAGC TATATGCACATGAAACCCATCCCGCCCAACCC CCTCAGC AAACCCATCCCGCCCAACCC GGGGGTTTTTT 7

Underline: binding region that hybridizes to target DNA; italics+bold: nicking endonuclease region

The above probes contain selective sequences (Table 2) previously verified by PCR for each gene detection. The PCR was performed for each target gene using the primers in Table 2, and the process of denaturing at 94°C for 20 seconds, annealing at 54°C for 20 seconds, and elongating at 72°C for 1 minute and 30 seconds was performed for 30 cycles, and then the generated sample was loaded onto a 2.5% agarose gel to confirm gene detection (Fig. 2).

Target
Food poisoning bacteria Target
gene Primer Sequence (5'-3') order
number E. coli O157:H7 VT2 Forward GGCACTGTCTGAAACTGTCC 8 Reverse TCGCCAGTTATCTGACATTCTG 9 Salmonella Typhimurium
(S ttr Forward ATTACAACATGGCTCACCAGGAG 10 Reverse GCTCAGACCAAAAGTGACCAT 11 Bacillus cereus hblD Forward ACCGGTAACACTATTCATGCATT 12 Reverse GGAGTCCATATGCTTAGATGCTG 13

Circular probes were prepared by ligating between the 3' and 5' ends of linear ssDNA in Table 1 using an ssDNA Ligation kit. The ligation reaction solution contained 15 ul of 50 uM padlock probe, 10 ul of 10X reaction buffer, 5 ul of 1 mM ATP, 5 ul of 50 mM MnCl ₂ , 4 ul of 100 U/ul CircLigase ssDNA Ligase, and 61 ul of distilled water (DW) in a total of 100 ul. The reaction solution was sequentially incubated at 60 °C for 6 h and at 80 °C for 10 min. Then, 120 units of exonuclease I and 20 units of exonuclease III were added to the reaction solution to remove residual linear ssDNA, and the reaction was sequentially incubated at 37 °C for 6 h and at 80 °C for 20 min. The resulting product was purified using the Oligo Clean-up and Concentration Kit, and verified by 15% denaturing urea-polyacrylamide gel electrophoresis with 1X TBE buffer at 100 V for 80 min, and gel stained with 1X SYBR Green I & II for 15 min. Afterwards, the gel was imaged using Alliance Mini HD9 (uvitec Cambridge, UK), and the fabrication of each circular probe was confirmed. That is, in order to produce a circular probe, single-stranded DNA and CircLigase and Exonulcease I & II were sequentially treated, and the step-by-step products were confirmed by electrophoresis, and the finally formed bands (CircLigase (+) and Exonulcease I & II (+)) represent the circular probe (Fig. 3).

Example 2: Circular probe-dependent RCA evaluation

To compare the amplification efficiency when only one type of circular probe was used and when a pair of circular probes were used, linear RCA was performed in the presence of genomic DNA of E. coli O157:H7 with i) VT2 F circular probe only, ii) VT2 R circular probe only, and iii) both VT2 F and VT2 R circular probes. After sonicating 30 ng/ul of genomic DNA for 20 min, hybridization reactions were performed sequentially at 95 °C for 5 min and at 54 °C for 10 min with a reaction solution containing 8 μl of VT2 circular probe (final concentration = 1.5 uM), 5 μl of 5X T4 ligase buffer, and 12 μl of genomic DNA.

After that, the reaction solution was mixed with 5 ul of 10X RCA Buffer, 5 ul of 10 mM dNTP, 3 ul of 10 U/ul phi29 DNA polymerase, and 12 ul of distilled water. 50 ul of the total reaction mixture for linear RCA was reacted at 30°C for 1 hour and inactivated at 65°C for 10 minutes. Meanwhile, PG-RCA was performed separately from linear RCA, and after preparing a reaction mixture containing the hybridization reaction solution as above (5 ul of 10X RCA Buffer, 5 ul of 10 mM dNTP, 3 ul of 10 U/ul phi29 DNA polymerase, 2 ul of NB.Bbvcl, and 10U distilled water), 50 ul of the reaction mixture was sequentially incubated at 30°C for 1 hour and at 65°C for 10 minutes. The generated samples were loaded onto a 15% denaturing urea-polyacrylamide gel with 1X TBE buffer at 180 V for 80 minutes, and stained with 1X SYBR Green I & II for 15 minutes. The gel was then imaged using an Alliance Mini HD9.

As a result, we confirmed that PG-RCA not only produces more DNA products than linear RCA, but also that PG-RCA using a pair of circular probes produces more DNA products than when only one type of probe is used (Fig. 4).

Example 3: Optimization of PG-RCA reaction

To optimize the PG-RCA reaction conditions, various samples were prepared by varying the concentrations of dNTP, phi29 DNA polymerase, or circular probe depending on the presence or absence of E. coli O157:H7 genomic DNA (indicated as C when absent or T when present). For samples containing genomic DNA, sonication was performed before the hybridization step. By sonication, DNA cleaved from genomic DNA can be easily obtained, thereby improving the yield of rolling circle amplification.

Hybridization reactions were performed under the same conditions as in Example 2 above with four concentrations (1.5 μM, 0.5 μM, 0.25 μM, 0.125 μM) of circular probes (VT2 F and VT2 R). Then, two different concentrations of dNTPs (0.1 mM, 1 mM) or four different concentrations (30 Units, 6 Units, 1.2 Units, 0.24 Units) of phi29 DNA polymerase were applied to the subsequent PG-RCA reaction. The PG-RCA reaction solution contained 25 μl of hybridization reaction solution, 5 μl of 10X RCA Buffer, 5 μl of dNTPs at various concentrations, 3 μl of phi29 DNA polymerase at various concentrations, 2 μl of NB.Bbvcl, and 10 μl of 5X SYBR Green II. The fluorescence values of the reaction solutions in the 96-well plate were measured at 30°C for 120 minutes at excitation and emission wavelengths of 480 nm and 535 nm at 30-s intervals using a microplate reader Sense Beta (HIDEX, Finland).

Under conditions using 1 mM dNTP, the fluorescence intensity of the target sample (T_phi29 30 U) increased faster than that of the negative control (w/o target DNA, C_phi29 30 U) when 30 U of phi29 DNA polymerase was used. On the other hand, when 6 U of phi29 DNA polymerase was used, the fluorescence intensity increase of the target sample was slower than when 30 U was used (Fig. 5).

When 30 U of phi29 DNA polymerase was used under the condition of 0.1 mM dNTP, the fluorescence intensity of the target sample (T_phi29 30 U) increased faster than that of the negative control (C_phi29 30 U), as in the previous case. However, when 6 U of phi29 DNA polymerase was used, it was confirmed that the signal amplification time of the target sample was much shorter when 0.1 mM dNTP was used than when 1 mM dNTP was used, which was more advantageous for target gene detection (Fig. 6).

Under two conditions of 30 U and 6 U of phi29 DNA polymerase, PG-RCA was performed using four concentrations (1.5, 0.5, 0.25, and 0.125 μM) of circular probe in the presence or absence of target genomic DNA. The threshold time difference between the target (T_phi29 6 U) and the control (C_phi29 6 U) was the largest when 1.5 μM circular probe was used in the condition of 6 U of phi29 DNA polymerase (Fig. 7), deriving the optimal conditions (dNTP 0.1 mM, phi29 DNA polymerase 6 U, circular probe 1.5 μM) for PG-RCA to become a more sensitive detection method.

Under the optimized conditions of PG-RCA, a control circular probe (con, SEQ ID NO: 7) containing a non-specific sequence was applied to three different genomic (E. coli O157:H7 (E), Salmonella Typhimurium (S), Bacillus cereus (B)) DNAs and the real-time PG-RCA results were compared with the samples with VT2-, ttr-, or hblD-specific circular probes. An increase in fluorescence intensity was observed in PG-RCA using VT2-, ttr-, and hblD-specific probes, but no increase in fluorescence intensity was observed when the control probe was used (Fig. 8).

These results indicate that probes containing nonspecific sequences that cannot hybridize to the target gene and random sequences excluding the hybridizing portion cannot interact with genomic DNA.

Example 4: Evaluation of PG-RCA according to target DNA concentration

To obtain the fluorescence intensity generated by PG-RCA, eight samples each of genomic DNA (E. coli O157:H7, Salmonella Typhimurium, Bacillus cereus) at concentrations of 0, 0.05, 0.2, 0.5, 1, 5, 10, and 20 ng/ul in the hybridization reaction solution with 1.5 μM of the circular probe were prepared. After sequentially incubating the samples at 95 °C for 5 min and at 54 °C for 10 min, the solutions were mixed with 5 μl of 10X RCA buffer, 5 μl of dNTP (0.1 mM), 3 μl of phi29 DNA polymerase (6 U), 2 μl of NB.Bbvcl, and 10 μl of 5X SYBR Green II. Afterwards, the fluorescence value of the reaction solution in the 96-well plate was measured at 1-minute intervals for 120 minutes at 30°C using a 7500 Real time PCR System (Thermo Fisher Scientific, MA, USA).

As a result, the threshold time decreased as the genomic DNA concentration increased, and the linear correlation between the threshold time and the DNA concentration was R2 = 0.9796 for E. coli O157:H7, R2 = 0.9781 for Salmonella Typhimurium, and R2 = 0.9583 for Bacillus cereus in the range of 0 to 1 ng/ul (Fig. 9 - A: Threshold time according to E. coli O157:H7 genomic DNA concentration, B: Threshold time according to Salmonella Typhimurium genomic DNA concentration, C: Threshold time according to Bacillus cereus genomic DNA concentration). These results suggest that the PG-RCA method has great potential to quantify target genomic DNA based on the standard curve obtained, and that there is an inverse correlation between DNA concentration and critical time in the range of 0 to 20 ng, and in particular, it shows linearity when the genomic DNA concentration is in the range of 0 to 1 ng/ul, suggesting that quantification is possible.

From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical idea or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of the present invention should be interpreted as including all changes or modifications derived from the meaning and scope of the patent claims described below rather than the above detailed description and their equivalent concepts within the scope of the present invention.

Attach electronic file of sequence list

Claims (15)

A method for detecting food poisoning bacteria comprising the following steps:
a) A step of sonicating the genomic DNA of food poisoning bacteria;
b) a step of hybridizing the sonicated genomic DNA and the circular probe pair;
c) a step of amplifying the target nucleic acid by mixing dNTP and DNA polymerase into the hybridized reaction product; and
d) A step of detecting the target nucleic acid.
In the first paragraph,
A detection method, wherein the above circular probe comprises a binding site complementary to a base sequence of 10 to 30 bp in a target nucleic acid.
In the first paragraph,
A detection method wherein the concentration of the above circular probe is 1 μM to 2 μM.
In the first paragraph,
A detection method wherein the concentration of the above dNTP is 0.05 mM to 0.2 mM.
In the first paragraph,
A detection method wherein the concentration of the above DNA polymerase is 2 U/ul to 10 U/ul.
In the first paragraph,
A method for detecting the food poisoning bacteria, wherein the food poisoning bacteria is at least one selected from the group consisting of Escherichia coli ( e. coli ), Salmonella Sp . , Bacillus Sp., Campylobacter Sp., Staphylococcus Sp., and Listeria Sp.
In Article 6,
A detection method wherein, when the food poisoning bacteria is Escherichia coli, the circular probe pair is a probe pair represented by sequence numbers 1 and 2.
In Article 6,
A detection method wherein, when the food poisoning bacteria is Salmonella genus, the circular probe pair is a probe pair represented by sequence numbers 3 and 4.
In Article 6,
A detection method wherein, when the food poisoning bacteria is of the genus Bacillus, the circular probe pair is a probe pair represented by sequence numbers 5 and 6.
In the first paragraph,
A detection method, wherein the step of amplifying the target nucleic acid is performed by primer generation rolling circle amplification (PG-RCA).
A composition for detecting food poisoning bacteria, comprising a pair of circular probes containing nucleic acid sequences complementary to food poisoning bacteria.
In Article 11,
A composition for detecting food poisoning bacteria, wherein when the food poisoning bacteria is Escherichia coli, the circular probe pair is a probe pair represented by sequence numbers 1 and 2, when the food poisoning bacteria is Salmonella genus, the circular probe pair is a probe pair represented by sequence numbers 3 and 4, and when the food poisoning bacteria is Bacillus genus, the circular probe pair is a probe pair represented by sequence numbers 5 and 6.
In Article 11,
The above composition is a composition for detecting food poisoning bacteria, comprising dNTP and DNA polymerase.
A kit for detecting food poisoning bacteria, comprising a composition according to any one of claims 11 to 13.
In Article 14,
The above kit is a kit for detecting food poisoning bacteria, which is a primer-forming rotary amplification kit.