CN111363860A

CN111363860A - Nucleic acid composition for detecting novel coronavirus COVID-19 and application

Info

Publication number: CN111363860A
Application number: CN202010458590.8A
Authority: CN
Inventors: 王笃强; 位小丫; 赵曼曼; 李瑞霞; 郑实
Original assignee: Wujiang Novoprotein Scientific Inc
Current assignee: Wujiang Novoprotein Scientific Inc
Priority date: 2020-05-27
Filing date: 2020-05-27
Publication date: 2020-07-03

Abstract

The invention discloses a nucleic acid composition for detecting novel coronavirus COVID-19 and application thereof, wherein a nucleic acid sample is expanded based on a transcription-mediated amplification Technology (TMA), and then the expanded virus target nucleic acid is specifically detected by combining the 'associated cleavage' activity of CRISPR-Cas13a protease. And adding the sgRNA, the Cas13a protein, the ssRNA signal report probe and a reaction buffer solution into a reaction system containing target nucleic acid to be detected, and reading and detecting signals of the sgRNA, the Cas13a protein, the ssRNA signal report probe and the reaction buffer solution when the reaction is carried out so as to detect the target gene. The method can be used for rapidly detecting whether a sample contains a target nucleic acid sequence and is combined with a transcription-mediated nucleic acid amplification technology, so that the sensitivity of the detection method can reach nanomolar level, and the method can be suitable for rapidly detecting pathogenic microorganisms, gene mutation, specific target RNA and the like.

Description

Nucleic acid composition for detecting novel coronavirus COVID-19 and application

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a nucleic acid composition for detecting a novel coronavirus COVID-19 and application thereof.

Background

The novel coronavirus COVID-19 can spread epidemic situation rapidly since the outbreak, does not draw the mind of Chinese people, bears unprecedented pressure on national epidemic situation prevention and control, and enters a public health event first-level response state in most provinces and cities. At present, there are 7 known human coronaviruses, HCoV-229E found in 1965, HCoV-OC43 identified in 1967, SARS-CoV appearing in the Guangdong of China in 2003, HCoV-NL63 isolated in the Netherlands in 2004, HCoV-HKU1 screened in hong Kong in 2005, MERS-CoV appearing in the middle east of 2012, and a novel coronaviruses COVID-19 found in Wuhan of China in 2019, respectively. The novel coronavirus COVID-19 is a transmissible infectious disease which causes clinical symptoms such as human respiratory diseases and severe pneumonia and is outbreaked in Wuhan city in Hubei province at the end of 12 months in 2019. By day 13 of 2/13 of 2020, 59885 people have been diagnosed nationwide as infected with the disease, 16067 in suspected cases, and 1368 die. The international committee for the classification of viruses (ICTV) states that the novel Coronavirus was named "SARS-Cov 2" (Severe acid respiratory syndrome corona 2). More recently, the name is "COVID-19". The coronavirus is coated with fat membrane, and the membrane surface contains three glycoproteins, namely spike glycoprotein, small envelope glycoprotein and membrane glycoprotein. The internal nucleic acid is single-stranded RNA with the length of 27-31kb, and has important structural characteristics specific to positive-strand RNA: namely, the 5 'end of the RNA chain has a methylated' hat 'structure, and the 3' end has a PolyA 'tail' structure, so that the function of a translation template can be exerted, and the transcription process of RNA-DNA-RNA is omitted. That is, the positive strand RNA of the genetic material in such viral particles typically enters the host cell and is directly translated as mRNA to the encoded protein, including the capsid protein and the viral RNA polymerase. RNA is replicated under the action of viral RNA replicase, dsRNA is synthesized by taking the RNA as a template, and the dsRNA is self-assembled into mature virus in the process of repeated replication and unwinding.

The nucleic acid detection is the most reliable screening and confirmation auxiliary means at present, and the positive diagnosis of the nucleic acid of samples such as sputum, throat swabs, lower respiratory tract secretions and the like is confirmed on the basis of meeting the suspected case standard. Because no effective vaccine against the virus is developed in the world at present, people in epidemic sites and related personnel can only be completely isolated for preventing the disease from spreading, and huge losses are brought to all aspects of production and life. Therefore, under the current severe prevention and control situation, there is a great need to develop a simple, rapid and highly sensitive differential diagnosis kit to provide a detection technology for clinical rapid diagnosis of infection of the novel coronavirus COVID-19 and other similar RNA viruses.

The specific nucleic acid or gene molecule detection method is one of the important advanced fields of modern medical development, and has application value, such as pathogen detection, genetic disease detection and the like. A method for diagnosing human body state and diseases by exploring the existence or change of unique characteristic nucleic acid molecule sequences of each pathogenic microorganism is also called Nucleic Acid Diagnostics (NADs). It is widely used in the fields of food safety, environmental microbial pollution detection, human pathogenic bacteria infection diagnosis and the like.

The prior art has the defects of long diagnosis time, complex operation, low sensitivity and the like. The complex and changeable diagnostic environment limits the application of gene molecule detection technology and simultaneously provides a challenge for the development of the technology. The acquired immune system of prokaryotes (such as bacteria and archaea) for resisting exogenous invading nucleic acids such as viruses is as follows: the CRISPR-Cas system brings revolutionary breakthrough in the technical field of gene editing. Among them, CRISPR-Cas9 specifically recognizes and cleaves a target sequence, and is widely developed as a genome editing tool. The 2015 seikovian group found that the endonuclease Cas12a, like Cas9 protein, was specifically cleaving target dsDNA guided by sgRNA or crRNA, except that Cas12a only required crRNA to guide specific cleavage of dsDNA, resulting in sticky ends for easy gene editing. The biggest difference is that Cas12a has the activity of 'bypass cutting' for non-specific arbitrary cutting of ssDNA after cutting target dsDNA, and according to the characteristic, a detection system for rapidly and accurately detecting virus DNA in samples such as cells, blood, saliva, urine, feces and the like based on the CRISPR-Cas12a system can be developed.

The target templates in the Cas9 and Cas12 detection systems related to the method are double-stranded DNA, and the current novel coronavirus COVID-19 nucleic acid is ssRNA. Zhang Feng et al in 2017 discovered a novel protein Cas13a, which is a VI type CRISPR-Cas system effector protein, has RNA mediated RNA enzyme cutting activity, and is the only protein capable of degrading RNA discovered by the second major class CRISPR-Cas system at present. In particular, Cas13a has a "tandem cleavage" (colateral effect) that can recognize and specifically cleave the target ssRNA under the guidance of sgRNA, and can cleave other non-target ssrnas (which can be designed as an RNA fluorescence reporter system) with unlimited rnase cleavage activity, and can detect target nucleic acids with specific sequences by virtue of this property. Zhang Feng et al utilize this special cleavage activity of the protein in combination with RPA technology (isothermal amplification) to establish a rapid nucleic acid detection technology, which is called SHERLOK (specific High Sensitivity Enzymatic Reporter UnLOCKing). Since the current situation of the novel coronavirus is very severe, the development of a novel coronavirus detection tool must ensure high sensitivity and strong fidelity, Cas13a-sgRNA can be activated by target RNA, so that high-sensitivity RNA detection in vitro is realized, and a rapid and accurate report result is also realized.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides a nucleic acid composition for detecting a novel coronavirus COVID-19 and application thereof. The detection kit prepared by the invention can perform transcription-mediated nucleic acid amplification technology under isothermal condition, rapidly expand the copy number of the viral RNA nucleic acid, and further improve the gene molecule detection technology by using a high specificity detection system which is used for cutting the viral nucleic acid by combining the CRISPR-Cas13a with a sgRNA system and further cutting ssRNA of a fluorescence quenching nucleic acid probe to generate a fluorescence signal, so that the virus can be accurately identified with trace amount, high sensitivity and high specificity. The technical means can not only rapidly identify the viruses such as novel coronavirus COVID-19, HIV, leukemia virus and other RNA viruses, but also be applied to various aspects such as disease screening and diagnosis, genetic disease, food sanitation detection, forensic identification, environmental pollution and the like.

In order to solve the technical problems, the invention provides the following technical scheme:

the first purpose of the invention provides a nucleic acid composition for detecting a novel coronavirus COVID-19, which comprises a primer pair N-t7F and N-R designed according to the N gene sequence of the novel coronavirus COVID-19 and a primer pair Orf1ab-t7F and Orf1ab-R designed according to the Orf1ab gene sequence of the novel coronavirus COVID-19, wherein the sequence of the N gene is shown as SEQ ID NO. 1, and the sequence of the Orf1ab gene is shown as SEQ ID NO. 2; the sequences of the primer pair N-t7F and N-R are shown as SEQ ID NO. 3 and SEQ ID NO. 4, and the sequences of the primer pair Orf1ab-t7F and Orf1ab-R are shown as SEQ ID NO. 5 and SEQ ID NO. 6.

Preferably, the kit further comprises an RNA nucleic acid probe, wherein the RNA nucleic acid probe is a chemically synthesized ssRNA nucleic acid probe with two ends subjected to fluorescent labeling modification, the sequence of the ssRNA nucleic acid probe is shown as SEQ ID NO. 9, the ssRNA nucleic acid probe is labeled with a fluorescent reporter group at the 5 'end, and is labeled with a fluorescent quenching group at the 3' end.

Preferably, the fluorescence reporter group is selected from one of HEX, Cy5, FAM and ROX, and the fluorescence quenching group is selected from one of BHQ1, BHQ3 and TAMRA.

The second purpose of the invention is to provide a kit for detecting the novel coronavirus COVID-19, wherein the kit comprises a nucleic acid composition for detecting the novel coronavirus COVID-19.

The third object of the present invention is to provide a method for detecting a novel coronavirus COVID-19 nucleic acid for non-disease diagnosis purposes, which comprises the following steps:

s1, chemically synthesizing an N gene and an Orf1ab gene fragment according to the sequence of the novel coronavirus COVID-19, artificially preparing a pseudovirus through transfection, namely a virus protein and nucleic acid compound, extracting nucleic acid, then diluting the pseudovirus in a gradient manner, and using the diluted pseudovirus as a transcription-mediated amplification template;

s2, designing a primer pair N-t7F and N-R according to the N gene sequence of the novel coronavirus COVID-19 and designing a primer pair Orf1ab-t7F and Orf1ab-R according to the Orf1ab gene sequence of the novel coronavirus COVID-19, and carrying out isothermal amplification on virus nucleic acid by using a transcription-mediated amplification system to expand the copy number of the virus nucleic acid; wherein, the sequence of the N gene is shown as SEQ ID NO. 1, and the sequence of the Orf1ab gene is shown as SEQ ID NO. 2; the sequences of the primer pair N-t7F and N-R are shown as SEQ ID NO. 3 and SEQ ID NO. 4, and the sequences of the primer pair Orf1ab-t7F and Orf1ab-R are shown as SEQ ID NO. 5 and SEQ ID NO. 6;

s3, chemically synthesizing a guided sgRNA of the Cas13a protein according to the N gene of the novel coronavirus COVID-19 and the cleavage target point of the Orf1ab gene sequence, wherein the sequence of the N-sgRNA corresponding to the N gene target point is shown as SEQ ID NO:7, and the sequence of the Orf1ab-sgRNA corresponding to the Orf1ab gene target point is shown as SEQ ID NO: 8;

s4, activating the non-specific RNase cleavage activity of the Cas13a protein after the CRISPR-Cas13a protein obtained by the in vitro recombinant expression method, the sgRNA cleavage system and the target nucleic acid form a 'ternary complex' system;

s5, constructing the ssRNA nucleic acid probe with both ends modified by the fluorescence reporter group and the fluorescence quencher group, wherein the activated Cas13a enzyme can generate a fluorescence signal when arbitrarily cutting the ssRNA nucleic acid probe, thereby quickly and accurately identifying and detecting the novel coronavirus COVID-19 nucleic acid.

Preferably, the specific process for artificially preparing the viral protein and nucleic acid complex in step S1 is as follows: cloning a target gene of the gene to a cell expression vector, transfecting cells after sequencing verification, then processing the cells to collect protein and nucleic acid compounds, removing genomic DNA residues by nuclease, extracting nucleic acid by a TRIZOL method, and calculating copy number to be used as a transcription-mediated amplification template.

Preferably, in step S2, 20 μ L of each component of the transcription-mediated amplification reaction system is gently vortexed and mixed uniformly, and then placed in a water bath at 42 ℃ for reaction for 15-25min to obtain the transcription product ssRNA, wherein the transcription-mediated amplification reaction system comprises the following components in the following concentrations: 2 mu.L of positive strand RNA fragment to be amplified, 1-5U RNase inhibitor, 500-2000U T7 RNA polymerase, 2000-4000U M-MLV reverse transcriptase, 0.5-2 mu M upstream primer, 0.5-2 mu M downstream primer and TMA reaction buffer; the positive strand RNA fragment to be amplified is the nucleic acid extracted in step S1;

wherein the TMA reaction buffer comprises the following components in the following concentrations: 20-50mM Tris-HCl pH8.0@25 ℃, 10-30mM KCl, 1-4mM MgCl₂1-5mM rNTPs, 1-5mM dNTPs, 20-50% glycerol, 0-10% DMSO and 0.5-1mM DTT.

Preferably, in step S4, the components of the ternary complex system are mixed by soft vortex, and then are subjected to heat preservation for 10-25min by a real-time fluorescence quantitative instrument at 37-40 ℃, and fluorescence is collected once every 40-60S of reaction, and is collected for 40-60min in a circulating manner; the "ternary complex" system comprises the following components in the following concentrations: RNA fragment to be detected, 0.5-2 muM TMA upstream primer, 0.5-2 muM TMA downstream primer, 2000-4000U M-MLV reverse transcriptase, 500-2000U T7 RNA polymerase, 1-5URNase inhibitor, 50-200nM cas13a protease, 50-400nM sgRNA, 0.25-1 muM RNA nucleic acid probe, 20-50mM Tris-HCl pH7.5@25 ℃, 20-40mM KCl, 1-5mM MgCl₂1-5mM rNTPs, 1-5mM dNTPs, 20-50% glycerol and 0.5-1mM DTT.

The invention has the beneficial effects that: the invention abandons the common PCR technology which firstly carries out reverse transcription on RNA into cDNA and needs the denaturation annealing process, and changes the transcription-mediated isothermal amplification virus nucleic acid amplification method into the detection technology which combines the CRISPR-Cas system, and has the advantages of faster reaction speed, more products, difficult pollution and higher fidelity.

In addition, the invention adopts CRISPR-Cas13a enzyme and sgRNA for specifically cutting novel coronavirus N and Orf1ab gene sequences, is different from the traditional virus detection technology, directly targets virus nucleic acid, can detect a virus nucleic acid sample with less than 100 copies in 60 minutes, and has strong specificity and high sensitivity.

Drawings

Fig. 1 is a technical schematic diagram of the combination of TMA and CRISPR-Cas13a of the present invention.

FIG. 2 is an SDS-PAGE electrophoresis of the Cas13a protein after ion exchange column purification.

FIG. 3 is a diagram showing the fluorescence collection result of the N gene fragment of the novel coronavirus COVID-19 of the present invention.

FIG. 4 is a graph showing the fluorescence collection result of the Orf1ab gene fragment of the novel coronavirus COVID-19 of the present invention.

FIG. 5 is a graph showing the fluorescence collection result of the Orf1ab gene fragment for the "two-in-one" detection of the novel coronavirus COVID-19 according to the present invention.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

First, as shown in FIG. 1, the upstream of the detection system was set to TMA technique, and under isothermal conditions, the target sequence was reverse transcribed under the action of wild-type M-MLV reverse transcriptase (containing RNase H activity) under the guide of a primer having a T7 promoter sequence added to the 5' end of the primer to form an RNA/DNA hybrid, and the RNase H activity of the reverse transcriptase degraded the RNA/DNA hybrid into ssDNA containing a promoter sequence recognized by T7 RNA polymerase. The other primer binds to the ssDNA and synthesizes dsDNA by reverse transcriptase. At this time, T7 RNA polymerase binds to the promoter and transcribes into ssRNA, which in turn can be used as a template for the next cycle, and the target sequence can be amplified for about 10-30min⁶In the above, the whole process is self-catalyzed in one tube. Secondly, a CRISPR-Cas13a detection system is used for designing sgRNA according to a target ssRNA target gene sequence by using a transcription product obtained by amplifying a nucleic acid fragment to be detected based on a TMA technology, wherein a signal report non-target ssRNA is required to be added into the detection system, and the signal amplification and reading can be realized by using the 'linkage cleavage' activity of Cas13a protein, so that the target gene is detected.

The technology related by the invention comprises the following steps:

(1) TRIZOL reagent method target ssRNA extraction (taking novel coronavirus COVID-19 as an example).

The specific extraction step is as follows: 1) taking out the pseudovirus from a refrigerator at the temperature of-80 ℃, melting in an ice bath or naturally melting at the temperature of 4 ℃, and then uniformly mixing; 2) 200ul of pseudovirus was mixed with 30ul of other unrelated RNA (10 ng/ul to 100 ng/ul), and then 800ul of TRIZOL reagent was added. Mixing gun heads uniformly, and standing for 5min at room temperature; 3) 160ul of chloroform was added in the step 2, and the mixture was inverted and mixed until the solution emulsified milky white. Standing at room temperature for 5 min. Centrifuging at 12000Xg for 15min, and taking out carefully; 4) the supernatant (approximately 700 ul) was aspirated and transferred to a new centrifuge tube. Adding 500ul isopropanol into the supernatant, turning upside down and mixing uniformly, and standing at room temperature for 20-30 min; 5) centrifuge at 12000Xg for 10 min. The tip was removed and the isopropanol carefully aspirated. Adding 800ul of 75% ethanol, washing upside down, and centrifuging at 12000Xg for 10 min; 6) taking out, pouring out 75% ethanol, air drying at room temperature for 5-10min, adding RNase Free water 50ul, and dissolving.

(2) And (2) carrying out amplification reaction on the RNA extracted in the step (1) by a transcription-mediated nucleic acid amplification technology.

The reagents of the reaction are respectively as follows: primers N-T7F and N-R, primers Orf1ab-T7F and Orf1ab-R, to-be-detected viral RNA, wild type M-MLV reverse transcriptase, T7 RNA polymerase, TMA reaction buffer and RNase Free water.

The Primer pair N-t7F and N-R, and the Primer pair Orf1ab-t7F and Orf1ab-R designed by the invention are respectively a pair of oligonucleotide single-stranded primers designed according to N and Orflab gene conserved sequences specific to the novel coronavirus COVID-19 by using Primer Premier 5.0 software and BLAST analysis of NCBI. It should be noted that, the two pairs of primers are respectively upstream and downstream of the specific recognition target region, the length is not more than 60nt, generally between 20-35nt, and the common problems of interference between primers, hairpin structure, primer dimer and the like are avoided. In addition, the 3 ' ends of the upstream primers N-t7F and Orf1ab-t7F can hybridize with the 3 ' end of the target gene to be detected, and the 5 ' end needs to be provided with a phage promoter sequence. The 3 'ends of the downstream primers N-R and Orf1ab-R are hybridized with the 3' end of the negative strand of the target gene to be detected.

Wherein, the T7 RNA polymerase can also be other phage RNA polymerases, such as T3, SP6 and the like. If other types of bacteriophage RNA polymerases are selected, the RNA polymerase binding recognition sequence on the 5' end of the upstream primer can be modified accordingly for subsequent in vitro transcription of the amplified product.

The wild type M-MLV reverse transcriptase is a murine reverse transcriptase without RNase H activity removal, and can also be other reverse transcriptases, such as AMV. If the added reverse transcriptase has no RNase H activity, RNase H can be additionally added into the system.

The transcription-mediated amplification reaction system in TMA technology mainly comprises RNase inhibitor, T7 RNA polymerase, M-MLV reverse transcriptase, upstream primer, downstream primer, Tris-HCl, ribonucleoside triphosphates (rNTPs), Dithiothreitol (DTT), deoxyribonucleoside triphosphates (dNTPs), KCl, MgCl and other components₂Glycerol and DMSO. The concentration of the concrete components is as follows: 20-50mM Tris-HCl pH8.0@25 ℃, 10-30mM KCl, 1-4mM MgCl₂1-5mM rNTPs, 1-5mM dNTPs, 20-50% glycerol, 0-10% DMSO, 0.5-1mM DTT, 0.5-2 μ M upstream primer, 0.5-2 μ M downstream primer and 1-5U RNase inhibitor, 2000-4000U M-MLV reverse transcriptase and 500-2000U T7 RNA polymerase.

The establishment of 20 μ l of amplification reaction system for the ssRNA of interest in the step (2): obtaining RNA of a related sample, fully and uniformly mixing the RNA with a certain concentration and the required components, and placing the reaction tube at 42 ℃ for reaction for 15-25min to obtain a transcription product ssRNA. Isothermal amplification reactions are generally carried out in simple isothermal metal baths or water bath chambers.

The virus may also be other similar RNA viruses. If other types of RNA viruses are selected, the upstream or downstream primers are designed according to the conserved region of the target virus.

(3) And (3) carrying out signal collection report on the transcription product in the step (2) by using a CRISPR-Cas13a detection system.

The reagents involved in the reaction of this step were respectively: cas protein, sgRNA, ssRNA of the transcription product, Cas reaction buffer, RNA nucleic acid probe and RNase Free water.

The Cas protein may be derived from different bacteria as Cas13a protein, such as one of LshCas13a (derived from Leptotrichia shahii), LwaCas13a (derived from leptorochia wadei) and LbuCas13a (derived from Leptotrichia buccalis), preferably LwaCas13 a.

The preservation buffer of the CRISPR-LwaCas13a protease comprises the following steps: 20mM Tris-HCl pH7.0@25 ℃, 0.1mM EDTA, 1mM DTT, 200mM NaCl, 50% (v/v) Glycerol.

The sgRNA according to step (3) refers to a guide RNA that comprises a target design for a gene of interest that can direct the Cas13a protein to specifically bind to a target ssRNA. The sgRNA is a structure of 5 '-simple repetitive sequence-recognition target sequence-3'; wherein, the Cas13a proteins of the same bacterial origin have the same simple repeat sequence, 5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC-3' is the simple repeat sequence of LwaCas13 a; the recognition target sequence is matched with a segment in the target RNA, and the length of the recognition target sequence is 25-38 nucleotides, and preferably more than 28. After the target point on the target gene is recognized by the Cas13a through the sgRNA, the RNase activity of the Cas13a can be activated, and the ssRNA nucleic acid probe is cut, so that a detection signal is obtained.

The RNA nucleic acid probe in the step (3) is a chemically synthesized ssRNA nucleic acid probe with two ends subjected to fluorescent labeling modification. The ssRNA nucleic acid probe is optimally labeled at the 5' end with a fluorescent reporter group, such as HEX, Cy5, FAM, ROX, etc., preferably FAM label; fluorescence quenching groups such as BHQ1, BHQ3, TAMRA, etc., preferably BHQ1, are optimally labeled at the 3' end. The detection method of the ssRNA nucleic acid probe is preferably a fluorescence detection method; the fluorescence detection method can be a method for detecting by using a microplate reader or a fluorescence spectrophotometer or a real-time fluorescence quantifier, and is preferably a real-time fluorescence quantifier. The sequence of the ssRNA nucleic acid probe of the invention is: 5 '[ FAM ] -GAAUUCCACCACGUUCCCGUGG-3' [ BHQ1], the sequence of which is shown as SEQ ID NO. 9.

The ssRNA nucleic acid probes can also be adapted to other experimental systems. While Cas13a protein-based cleavage of target RNA specifically under sgRNA guidance, rnase activity of Cas13a protease can degrade any ssRNA that carries a signal, thereby releasing a fluorescent signal.

The establishment of 20 μ L CRISPR-Cas13a protease cleavage system of step (3) is reported for signal collection: 0.25-10 μ M Cas13a protease, 50-400nM sgRNA (sgRNA: Cas13a protein molarity between 1:1-1: 4), 500nM RNA nucleic acid probe, 2 μ L TMA transcript (1-100ng) and Cas reaction buffer to a final concentration of 20mM Tris-HCl pH7.0@25 ℃, 30mM KCL and 2.5mM MgCl 2. Collecting fluorescence every 40-60s by a fluorescence quantitative instrument at 37-42 deg.C for 30-60 min.

And (3) judging the result of the step (3), wherein the reported positive signal is a fluorescent signal based on the RNase activity of the Cas13a protein, and the positive fluorescent signal is detected by adding an ssRNA nucleic acid probe with two ends connected with a fluorescent substance and a quencher into the system, so that after the Cas13a protease recognizes the target RNA with the target sequence with the help of sgRNA, the activated RNase activity can degrade the RNA with the signal, thereby releasing the fluorescent signal.

Taking the fluorescence signal as an example, the following formula should be calculated:

the positive judgment basis is as follows: f_{Final experimental group}-F_{Experimental groups the first}>(F_{Negative group Final}-F_{Negative group first})x100

Wherein F represents a fluorescence signal value. The negative control group is a group of negative signals corresponding to each experimental group without the addition of the target gene of RNA.

The method provided by the invention is used for qualitative determination, so that the final fluorescent signal can be judged to be positive if the final fluorescent signal is more than 100 times higher than that of a negative sample, and can be judged to be strong positive if the final fluorescent signal is more than 1000 times higher than that of the negative sample; if the sample is 10 times to 100 times higher than the negative sample, the sample input amount can be increased for retesting.

The present invention does not require complicated temperature control equipment or system, and moreover, the above two steps can be preferably combined in one reaction system, thereby simplifying the operation process.

"two-in-one" preferred reaction system (50 μ L): RNA fragment to be detected, TMA upstream primer (0.5-2 μ M), TMA downstream primer (0.5-2 μ M), 2000-4000U M-MLV reverse transcriptase, 500-2000U T7 RNA polymerase, 1-5U RNase inhibitor, 50-200nM cas13a protease, 50-400nM sgRNA, 0.25-1 μ M RNA nucleic acid probe, final concentration of 20-50mM Tris-HCl pH7.5@25 ℃, 20-40mM KCl, 1-5mM MgCl₂1-5mM rNTPs, 1-5mM dNTPs, 20-50% glycerol and 0.5-1mM DTT. The reaction tube is firstly processed by a real-time fluorescence quantitative instrument at the temperature of 37-40 DEG CAfter the temperature is kept for 10-25min, fluorescence is collected once every 40-60s of reaction, and the fluorescence is collected for 40-60min in a circulating way.

The final detection means and positive judgment method are basically the same as those of the two-step separation reaction system.

The invention provides a detection technology for accurately identifying viruses after the TMA transcription mediated amplification system is used together with the CRISPR-Cas13a protein editing system, and the detection technology has higher practical value.

The invention also provides a ssRNA nucleic acid probe designed by utilizing the non-specific RNA enzyme activity in the CRISPR-Cas13a protein editing system, and the nucleic acid probe can be used for detection reactions of different targets and has universality.

Example 1

The present embodiment provides a method for detecting N gene of novel coronavirus COVID-19, comprising the following steps:

firstly, in order to detect the expression of the novel coronavirus RNA, the nucleotide sequence of the N gene of the novel coronavirus COVID-19 is searched according to NCBI and Genbank databases, a section of N gene conserved sequence of the novel coronavirus COVID-19 is chemically synthesized and transfected into cells, pseudoviruses are artificially prepared, gradient dilution is carried out after extraction, and the diluted RNA is used as a template of transcription-mediated amplification (TMA).

Second, the upstream and downstream primers of the N gene for TMA amplification of COVID-19 were designed and synthesized by selecting a region free of secondary structure and highly conserved. Wherein the sequence of the upstream primer N-t7F is as follows: 5'-AATTTAATACGACTCACTATAGGGtgtaggtcaaccacgttccc-3' are provided. Wherein the capital letters are T7 promoter sequences and the small letters are RNA (+) binding sequences. The downstream primer N-R is as follows: 5'-caactccaggcagcagtagg-3', the downstream primer is a sequence that hybridizes to RNA (-).

Thirdly, by designing and synthesizing a primer, the N-sgRNA targeting the RNA minus strand fragment in the above step is synthesized by using a boiling water annealing primer and an in vitro transcription method, and the sequence is as follows: 5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACACUAAAGCAUACAAUGUAACACAAGCUUUC-3' are provided. Wherein the sequence format of the N-sgRNA is as follows: the 5 '-Cas 13a protein-binding simple repeat (36 nt) + matches the fragment in the targeting RNA (-) by (30 nt) -3'.

Fourth, a TMA reaction system (20 μ L) was constructed, maintaining the following components at the following concentrations: 2 μ L of the positive strand RNA fragment to be amplified, T7 RNA polymerase (2000U), M-MLV reverse transcriptase (4000U), RNase inhibitor (2U), the above upstream primer N-T7F (0.5 μ M) and downstream primer N-R (0.5 μ M), and TMA reaction buffer (20-50 mM Tris-HCl pH8.0@25 ℃, 10-30mM KCl, 1-4mM MgCl₂1-5mM rNTPs, 1-5mM dNTPs, 20-50% glycerol, 0-10% DMSO, 0.5-1mM DTT). After the components of the reaction system are gently swirled and mixed uniformly, the mixture is placed in a water bath kettle for reaction at 42 ℃ for 15-25min to obtain a transcription product, and the product is the negative strand RNA of the positive strand RNA to be amplified.

Fifth, nucleic acid sequence signal reporting is performed based on the establishment of a unique target RNA-activated "strand-cleavage" non-specific rnase activity of the Cas13a protein. The concentrations in the reagents participating in the reaction were: the transcription products ssRNA (10-100 ng) of the TMA technique described above, LwaCas13a protease (1. mu.M), N-sgRNA (400 nM), cas buffer (20mM Tris-HCl pH7.0@25 ℃, 50mM KCL, 2.5mM MgCl₂) 500nM OligoRNA-Cas13a-probe (5 'FAM label and 3' BHQ1 quencher label) and RNase Free Water. And (3) after the components of the reaction system are mixed evenly in a soft vortex mode, the mixture is placed in a real-time fluorescence quantitative instrument to be set at 37 ℃, a fluorescence signal is collected every 40-60s of reaction, and the collection is carried out for 45-100 times in a circulating mode.

The negative control group is a set of negative signals corresponding to each experimental group without addition of the transcription product ssRNA (RNase Free Water complement system).

The detection result shows that the sample of the experimental group can detect the fluorescence data and the fluorescence data also obviously rises along with the increase of the detection time, while the negative control group only detects the background fluorescence but the fluorescence data does not increase along with the detection time. And judging the experimental group as a positive sample according to the positive judgment formula.

The experimental results show that the detection system disclosed in the present invention can detect the N gene fragment detecting the novel coronavirus COVID-19 as low as about 10nM (as shown in FIG. 3) compared to the control group (the negative control is RNase Free Water). FIG. 3 is a graph showing the fluorescence collection results of the N gene fragment of the novel coronavirus COVID-19 of the present invention, wherein 1 in FIG. 3 represents 1. mu. mol of ssRNA, 2 represents 100mmol of ssRNA, 3 represents 10mmol of ssRNA, 4 represents 1mmol of ssRNA, 5 represents 100 nmolsRNA, 6 represents 10nmol of ssRNA, and 7 represents 0 ssRNA (negative control).

Example 2

The embodiment provides a method for rapidly detecting and detecting Orf1ab gene of novel coronavirus COVID-19, which comprises the following steps:

first, pseudoviruses were artificially prepared as templates for transcription-mediated amplification (TMA).

Second, upstream and downstream primers for TMA amplification of the Orf1ab gene of COVID-19 were designed and synthesized. Wherein the upstream primer Orf1ab-t7F is: 5'-AATTTAATACGACTCACTATAGGGgcattctgtgaattataagg-3' are provided. Wherein, 24 bases at the 5 ' end of Orf1ab-T7F are the added T7 phage promoter sequence, and 20 bases at the 3 ' end are the specific sequence hybridized with the (+) 5 ' end of the template RNA. The downstream primer Orf1ab-R is: 5'-atactgctgccgtgaacatg-3', is a sequence that hybridizes to RNA (-).

Thirdly, sgRNA targeting the RNA minus strand fragment is synthesized by designing and synthesizing a primer, and utilizing a boiling water annealing primer and an in vitro transcription method, and the sequence is as follows: 5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUUUGUGUGCUGACUCUAUCAUUAUUGGU-3' are provided. Wherein the sequence format of Orf1ab-sgRNA is as follows: the 5 '-Cas 13a protein-bound simple repeat (36 nt) + hybridizes to the fragment in the targeting RNA (-) (28 nt) -3'.

Fourth, reference example 1 was used to extract pseudoviral RNA and construct a TMA reaction system (20. mu.L). After the components of the reaction system are gently swirled and mixed uniformly, the mixture is placed in a water bath kettle for reaction at 42 ℃ for 15-25min to obtain a transcription product, and the product is the negative strand RNA of the positive strand RNA to be amplified.

Fifth, reference example 1 establishes a nucleic acid sequence signal reporting reaction system based on the unique targeted RNA-activated rnase activity of Cas13a protein. The concentrations in the reagents participating in the reaction were: the transcription products ssRNA (10-100 ng) of the TMA technique described above, LwaCas13a protease (4. mu.M), Orf1ab-sgRNA (100 nM), cas buffer (10mM Tris-HClpH7.0@25 ℃, 30mM KCL, 2.5mM MgCl₂)，500nM OligoRNA-Cas13a-probe and RNase FreeWater. And (3) uniformly mixing the reaction system, placing the mixture in a real-time fluorescence quantitative instrument to set the temperature of 37-42 ℃, collecting a fluorescence signal every 50s of reaction, and circularly collecting for 45-100 times.

The detection result shows that the fluorescence data detected by the experimental group and gradually increased along with the increase of the detection time, while the fluorescence data only detected by the negative control group but did not increase along with the detection time. And judging whether the sample is a positive sample according to the positive judgment formula.

The experimental results show that compared with the control group (the negative control is Rnase Free Water), the system based on the TMA technology combined with CRISPR-Cas13a disclosed in the present invention can detect the Orf1ab gene fragment of the human novel coronavirus COVID-19 (fig. 4) as low as about 10 nM. FIG. 4 is a graph showing the fluorescence collection result of the Orf1ab gene fragment of the novel coronavirus COVID-19 of the present invention. In FIG. 4, 1 represents 1. mu. mol of ssRNA, 2 represents 100mmol of ssRNA, 3 represents 10mmol of ssRNA, 4 represents 1mmol of ssRNA, 5 represents 100nmol of ssRNA, 6 represents 10nmol of ssRNA, and 7 represents 0 ssRNA (negative control).

Example 3

The embodiment provides a method for detecting Orf1ab gene of novel coronavirus COVID-19 by combining TMA and Cas13a, which comprises the following steps:

first, the template, primer and nucleic acid probe required in said example 3 can be prepared according to example 2.

Second, a TMA in combination with Cas13a "two-in-one" reaction system (50 μ L) was established. The reagents and specific concentrations involved in the reaction were: RNA (+) fragments to be detected, TMA upstream and downstream primers Orf1ab-t7F (1. mu.M) and Orf1ab-R (1. mu.M), 2000-4000U M-MLV reverse transcriptase, 500-2000U T7 RNA polymerase, 4U RNase inhibitor, 2. mu.M Cas13a protease, 400nM Orf1ab-sgRNA, 0.5. mu.M OligoRNA-Cas13a-probe, TC reaction buffer (20-50 mM Tris-HClpH7.5@25 ℃, 20-40mM KCl, 1-5mM MgCl₂1-5mM rNTPs, 1-5mM dNTPs, 20-50% glycerol and 0.5-1mM DTT).

Thirdly, after the components of the reaction system are mixed evenly by soft vortex, the reaction tube is reacted for 10 to 25min by a fluorescence quantitative instrument at the temperature of between 37 and 42 ℃, fluorescence is collected once every 40 to 60s of reaction at the temperature of between 37 ℃ and is collected for 45 to 100 times in a circulating way.

The negative control group is a set of negative signals corresponding to each experimental group without template RNA addition (RNase Free Water complement system addition).

The experimental results show that compared with the control group (the negative control is Rnase Free Water), the system based on the TMA and CRISPR-Cas13a two-in-one combination can detect the Orf1ab gene fragment of the novel coronavirus COVID-19 (shown in FIG. 5) as low as about 10 nM. FIG. 5 is a graph showing the fluorescence collection result of the Orf1ab gene fragment for the "two-in-one" detection of the novel coronavirus COVID-19 according to the present invention. In FIG. 5, 1 represents 1. mu. mol of ssRNA, 2 represents 100mmol of ssRNA, 3 represents 10mmol of ssRNA, 4 represents 1mmol of ssRNA, 5 represents 100nmol of ssRNA, 6 represents 10nmol of ssRNA, 7 represents 1nmol of ssRNA, and 8 represents 0 ssRNA (negative control).

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Wujiang Yoashan protein science and technology Co Ltd

<120> nucleic acid composition for detecting novel coronavirus COVID-19 and application thereof

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augugcuaug aggcccaauu ucacuauuaa ggguucauuc cuuaaugguu caugugguag 10020

uguugguuuu aacauagauu augacugugu cucuuuuugu uacaugcacc auauggaauu 10080

accaacugga guucaugcug gcacagacuu agaagguaac uuuuauggac cuuuuguuga 10140

caggcaaaca gcacaagcag cugguacgga cacaacuauu acaguuaaug uuuuagcuug 10200

guuguacgcu gcuguuauaa auggagacag gugguuucuc aaucgauuua ccacaacucu 10260

uaaugacuuu aaccuugugg cuaugaagua caauuaugaa ccucuaacac aagaccaugu 10320

ugacauacua ggaccucuuu cugcucaaac uggaauugcc guuuuagaua ugugugcuuc 10380

auuaaaagaa uuacugcaaa augguaugaa uggacguacc auauugggua gugcuuuauu 10440

agaagaugaa uuuacaccuu uugauguugu uagacaaugc ucagguguua cuuuccaaag 10500

ugcagugaaa agaacaauca aggguacaca ccacugguug uuacucacaa uuuugacuuc 10560

acuuuuaguu uuaguccaga guacucaaug gucuuuguuc uuuuuuuugu augaaaaugc 10620

cuuuuuaccu uuugcuaugg guauuauugc uaugucugcu uuugcaauga uguuugucaa 10680

acauaagcau gcauuucucu guuuguuuuu guuaccuucu cuugccacug uagcuuauuu 10740

uaauaugguc uauaugccug cuaguugggu gaugcguauu augacauggu uggauauggu 10800

ugauacuagu uugucugguu uuaagcuaaa agacuguguu auguaugcau cagcuguagu 10860

guuacuaauc cuuaugacag caagaacugu guaugaugau ggugcuagga gaguguggac 10920

acuuaugaau gucuugacac ucguuuauaa aguuuauuau gguaaugcuu uagaucaagc 10980

cauuuccaug ugggcucuua uaaucucugu uacuucuaac uacucaggug uaguuacaac 11040

ugucauguuu uuggccagag guauuguuuu uauguguguu gaguauugcc cuauuuucuu 11100

cauaacuggu aauacacuuc aguguauaau gcuaguuuau uguuucuuag gcuauuuuug 11160

uacuuguuac uuuggccucu uuuguuuacu caaccgcuac uuuagacuga cucuuggugu 11220

uuaugauuac uuaguuucua cacaggaguu uagauauaug aauucacagg gacuacuccc 11280

acccaagaau agcauagaug ccuucaaacu caacauuaaa uuguugggug uugguggcaa 11340

accuuguauc aaaguagcca cuguacaguc uaaaauguca gauguaaagu gcacaucagu 11400

agucuuacuc ucaguuuugc aacaacucag aguagaauca ucaucuaaau ugugggcuca 11460

auguguccag uuacacaaug acauucucuu agcuaaagau acuacugaag ccuuugaaaa 11520

aaugguuuca cuacuuucug uuuugcuuuc caugcagggu gcuguagaca uaaacaagcu 11580

uugugaagaa augcuggaca acagggcaac cuuacaagcu auagccucag aguuuaguuc 11640

ccuuccauca uaugcagcuu uugcuacugc ucaagaagcu uaugagcagg cuguugcuaa 11700

uggugauucu gaaguuguuc uuaaaaaguu gaagaagucu uugaaugugg cuaaaucuga 11760

auuugaccgu gaugcagcca ugcaacguaa guuggaaaag auggcugauc aagcuaugac 11820

ccaaauguau aaacaggcua gaucugagga caagagggca aaaguuacua gugcuaugca 11880

gacaaugcuu uucacuaugc uuagaaaguu ggauaaugau gcacucaaca acauuaucaa 11940

caaugcaaga gaugguugug uucccuugaa cauaauaccu cuuacaacag cagccaaacu 12000

aaugguuguc auaccagacu auaacacaua uaaaaauacg ugugauggua caacauuuac 12060

uuaugcauca gcauuguggg aaauccaaca gguuguagau gcagauagua aaauuguuca 12120

acuuagugaa auuaguaugg acaauucacc uaauuuagca uggccucuua uuguaacagc 12180

uuuaagggcc aauucugcug ucaaauuaca gaauaaugag cuuaguccug uugcacuacg 12240

acagaugucu ugugcugccg guacuacaca aacugcuugc acugaugaca augcguuagc 12300

uuacuacaac acaacaaagg gagguagguu uguacuugca cuguuauccg auuuacagga 12360

uuugaaaugg gcuagauucc cuaagaguga uggaacuggu acuaucuaua cagaacugga 12420

accaccuugu agguuuguua cagacacacc uaaagguccu aaagugaagu auuuauacuu 12480

uauuaaagga uuaaacaacc uaaauagagg uaugguacuu gguaguuuag cugccacagu 12540

acgucuacaa gcugguaaug caacagaagu gccugccaau ucaacuguau uaucuuucug 12600

ugcuuuugcu guagaugcug cuaaagcuua caaagauuau cuagcuagug ggggacaacc 12660

aaucacuaau uguguuaaga uguuguguac acacacuggu acuggucagg caauaacagu 12720

uacaccggaa gccaauaugg aucaagaauc cuuugguggu gcaucguguu gucuguacug 12780

ccguugccac auagaucauc caaauccuaa aggauuuugu gacuuaaaag guaaguaugu 12840

acaaauaccu acaacuugug cuaaugaccc uguggguuuu acacuuaaaa acacagucug 12900

uaccgucugc gguaugugga aagguuaugg cuguaguugu gaucaacucc gcgaacccau 12960

gcuucaguca gcugaugcac aaucguuuuu aaacggguuu gcgguguaag ugcagcccgu 13020

cuuacaccgu gcggcacagg cacuaguacu gaugucguau acagggcuuu ugacaucuac 13080

aaugauaaag uagcugguuu ugcuaaauuc cuaaaaacua auuguugucg cuuccaagaa 13140

aaggacgaag augacaauuu aauugauucu uacuuuguag uuaagagaca cacuuucucu 13200

aacuaccaac augaagaaac aauuuauaau uuacuuaagg auuguccagc uguugcuaaa 13260

caugacuucu uuaaguuuag aauagacggu gacaugguac cacauauauc acgucaacgu 13320

cuuacuaaau acacaauggc agaccucguc uaugcuuuaa ggcauuuuga ugaagguaau 13380

ugugacacau uaaaagaaau acuugucaca uacaauuguu gugaugauga uuauuucaau 13440

aaaaaggacu gguaugauuu uguagaaaac ccagauauau uacgcguaua cgccaacuua 13500

ggugaacgug uacgccaagc uuuguuaaaa acaguacaau ucugugaugc caugcgaaau 13560

gcugguauug uugguguacu gacauuagau aaucaagauc ucaaugguaa cugguaugau 13620

uucggugauu ucauacaaac cacgccaggu aguggaguuc cuguuguaga uucuuauuau 13680

ucauuguuaa ugccuauauu aaccuugacc agggcuuuaa cugcagaguc acauguugac 13740

acugacuuaa caaagccuua cauuaagugg gauuuguuaa aauaugacuu cacggaagag 13800

agguuaaaac ucuuugaccg uuauuuuaaa uauugggauc agacauacca cccaaauugu 13860

guuaacuguu uggaugacag augcauucug cauugugcaa acuuuaaugu uuuauucucu13920

acaguguucc caccuacaag uuuuggacca cuagugagaa aaauauuugu ugaugguguu 13980

ccauuuguag uuucaacugg auaccacuuc agagagcuag guguuguaca uaaucaggau 14040

guaaacuuac auagcucuag acuuaguuuu aaggaauuac uuguguaugc ugcugacccu 14100

gcuaugcacg cugcuucugg uaaucuauua cuagauaaac gcacuacgug cuuuucagua 14160

gcugcacuua cuaacaaugu ugcuuuucaa acugucaaac ccgguaauuu uaacaaagac 14220

uucuaugacu uugcuguguc uaaggguuuc uuuaaggaag gaaguucugu ugaauuaaaa 14280

cacuucuucu uugcucagga ugguaaugcu gcuaucagcg auuaugacua cuaucguuau 14340

aaucuaccaa caauguguga uaucagacaa cuacuauuug uaguugaagu uguugauaag 14400

uacuuugauu guuacgaugg uggcuguauu aaugcuaacc aagucaucgu caacaaccua 14460

gacaaaucag cugguuuucc auuuaauaaa ugggguaagg cuagacuuua uuaugauuca 14520

augaguuaug aggaucaaga ugcacuuuuc gcauauacaa aacguaaugu caucccuacu 14580

auaacucaaa ugaaucuuaa guaugccauu agugcaaaga auagagcucg caccguagcu 14640

ggugucucua ucuguaguac uaugaccaau agacaguuuc aucaaaaauu auugaaauca 14700

auagccgcca cuagaggagc uacuguagua auuggaacaa gcaaauucua uggugguugg 14760

cacaacaugu uaaaaacugu uuauagugau guagaaaacc cucaccuuau ggguugggau 14820

uauccuaaau gugauagagc caugccuaac augcuuagaa uuauggccuc acuuguucuu 14880

gcucgcaaac auacaacgug uuguagcuug ucacaccguu ucuauagauu agcuaaugag 14940

ugugcucaag uauugaguga aauggucaug uguggcgguu cacuauaugu uaaaccaggu 15000

ggaaccucau caggagaugc cacaacugcu uaugcuaaua guguuuuuaa cauuugucaa 15060

gcugucacgg ccaauguuaa ugcacuuuua ucuacugaug guaacaaaau ugccgauaag 15120

uauguccgca auuuacaaca cagacuuuau gagugucucu auagaaauag agauguugac 15180

acagacuuug ugaaugaguu uuacgcauau uugcguaaac auuucucaau gaugauacuc 15240

ucugacgaug cuguugugug uuucaauagc acuuaugcau cucaaggucu aguggcuagc 15300

auaaagaacu uuaagucagu ucuuuauuau caaaacaaug uuuuuauguc ugaagcaaaa 15360

uguuggacug agacugaccu uacuaaagga ccucaugaau uuugcucuca acauacaaug 15420

cuaguuaaac agggugauga uuauguguac cuuccuuacc cagauccauc aagaauccua 15480

ggggccggcu guuuuguaga ugauaucgua aaaacagaug guacacuuau gauugaacgg 15540

uucgugucuu uagcuauaga ugcuuaccca cuuacuaaac auccuaauca ggaguaugcu 15600

gaugucuuuc auuuguacuu acaauacaua agaaagcuac augaugaguu aacaggacac 15660

auguuagaca uguauucugu uaugcuuacu aaugauaaca cuucaaggua uugggaaccu 15720

gaguuuuaug aggcuaugua cacaccgcau acagucuuac aggcuguugg ggcuuguguu 15780

cuuugcaauu cacagacuuc auuaagaugu ggugcuugca uacguagacc auucuuaugu 15840

uguaaaugcu guuacgacca ugucauauca acaucacaua aauuagucuu gucuguuaau 15900

ccguauguuu gcaaugcucc agguugugau gucacagaug ugacucaacu uuacuuagga 15960

gguaugagcu auuauuguaa aucacauaaa ccacccauua guuuuccauu gugugcuaau 16020

ggacaaguuu uugguuuaua uaaaaauaca uguguuggua gcgauaaugu uacugacuuu 16080

aaugcaauug caacauguga cuggacaaau gcuggugauu acauuuuagc uaacaccugu 16140

acugaaagac ucaagcuuuu ugcagcagaa acgcucaaag cuacugagga gacauuuaaa 16200

cugucuuaug guauugcuac uguacgugaa gugcugucug acagagaauu acaucuuuca 16260

ugggaaguug guaaaccuag accaccacuu aaccgaaauu augucuuuac ugguuaucgu 16320

guaacuaaaa acaguaaagu acaaauagga gaguacaccu uugaaaaagg ugacuauggu 16380

gaugcuguug uuuaccgagg uacaacaacu uacaaauuaa auguugguga uuauuuugug 16440

cugacaucac auacaguaau gccauuaagu gcaccuacac uagugccaca agagcacuau 16500

guuagaauua cuggcuuaua cccaacacuc aauaucucag augaguuuuc uagcaauguu 16560

gcaaauuauc aaaagguugg uaugcaaaag uauucuacac uccagggacc accugguacu 16620

gguaagaguc auuuugcuau uggccuagcu cucuacuacc cuucugcucg cauaguguau 16680

acagcuugcu cucaugccgc uguugaugca cuaugugaga aggcauuaaa auauuugccu 16740

auagauaaau guaguagaau uauaccugca cgugcucgug uagaguguuu ugauaaauuc 16800

aaagugaauu caacauuaga acaguauguc uuuuguacug uaaaugcauu gccugagacg 16860

acagcagaua uaguugucuu ugaugaaauu ucaauggcca caaauuauga uuugaguguu 16920

gucaaugcca gauuacgugc uaagcacuau guguacauug gcgacccugc ucaauuaccu 16980

gcaccacgca cauugcuaac uaagggcaca cuagaaccag aauauuucaa uucagugugu 17040

agacuuauga aaacuauagg uccagacaug uuccucggaa cuugucggcg uuguccugcu 17100

gaaauuguug acacugugag ugcuuugguu uaugauaaua agcuuaaagc acauaaagac 17160

aaaucagcuc aaugcuuuaa aauguuuuau aaggguguua ucacgcauga uguuucaucu 17220

gcaauuaaca ggccacaaau aggcguggua agagaauucc uuacacguaa cccugcuugg 17280

agaaaagcug ucuuuauuuc accuuauaau ucacagaaug cuguagccuc aaagauuuug 17340

ggacuaccaa cucaaacugu ugauucauca cagggcucag aauaugacua ugucauauuc 17400

acucaaacca cugaaacagc ucacucuugu aauguaaaca gauuuaaugu ugcuauuacc 17460

agagcaaaag uaggcauacu uugcauaaug ucugauagag accuuuauga caaguugcaa 17520

uuuacaaguc uugaaauucc acguaggaau guggcaacuu uacaagcuga aaauguaaca 17580

ggacucuuua aagauuguag uaagguaauc acuggguuac auccuacaca ggcaccuaca 17640

caccucagug uugacacuaa auucaaaacu gaagguuuau guguugacau accuggcaua 17700

ccuaaggaca ugaccuauag aagacucauc ucuaugaugg guuuuaaaau gaauuaucaa 17760

guuaaugguu acccuaacau guuuaucacc cgcgaagaag cuauaagaca uguacgugca 17820

uggauuggcu ucgaugucga ggggugucau gcuacuagag aagcuguugg uaccaauuua 17880

ccuuuacagc uagguuuuuc uacagguguu aaccuaguug cuguaccuac agguuauguu 17940

gauacaccua auaauacaga uuuuuccaga guuagugcua aaccaccgcc uggagaucaa 18000

uuuaaacacc ucauaccacu uauguacaaa ggacuuccuu ggaauguagu gcguauaaag 18060

auuguacaa 18069

<210>3

<211>44

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

aatttaatac gactcactat agggtgtagg tcaaccacgt tccc 44

<210>4

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

caactccagg cagcagtagg 20

<210>5

<211>44

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

aatttaatac gactcactat aggggcattc tgtgaattat aagg 44

<210>6

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

atactgctgc cgtgaacatg 20

<210>7

<211>66

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

gauuuagacu accccaaaaa cgaaggggac uaaaacacua aagcauacaa uguaacacaa 60

gcuuuc 66

<210>8

<211>64

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gauuuagacu accccaaaaa cgaaggggac uaaaacuuug ugugcugacu cuaucauuau 60

uggu 64

<210>9

<211>22

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

gaauuccacc acguucccgu gg 22

Claims

1. A nucleic acid composition for detecting a novel coronavirus COVID-19 is characterized by comprising a primer pair N-t7F and N-R designed according to the N gene sequence of the novel coronavirus COVID-19 and a primer pair Orf1ab-t7F and Orf1ab-R designed according to the Orf1ab gene sequence of the novel coronavirus COVID-19, wherein the sequence of the N gene is shown as SEQ ID NO. 1, and the sequence of the Orf1ab gene is shown as SEQ ID NO. 2; the sequences of the primer pair N-t7F and N-R are shown as SEQ ID NO. 3 and SEQ ID NO. 4, and the sequences of the primer pair Orf1ab-t7F and Orf1ab-R are shown as SEQ ID NO. 5 and SEQ ID NO. 6.

2. The nucleic acid composition for detecting the novel coronavirus COVID-19 according to claim 1, further comprising an RNA nucleic acid probe, wherein the RNA nucleic acid probe is a chemically synthesized ssRNA nucleic acid probe with two ends subjected to fluorescent labeling modification, the sequence of the ssRNA nucleic acid probe is shown as SEQ ID NO. 9, the ssRNA nucleic acid probe is labeled with a fluorescent reporter group at the 5 'end and a fluorescent quencher group at the 3' end.

3. The nucleic acid composition for detecting the novel coronavirus COVID-19 according to claim 2, wherein the fluorescent reporter group is selected from one of HEX, Cy5, FAM and ROX, and the fluorescent quencher group is selected from one of BHQ1, BHQ3 and TAMRA.

4. A kit for detecting a novel coronavirus covi-19, wherein the kit comprises the nucleic acid composition for detecting a novel coronavirus covi-19 according to any one of claims 1 to 3.

5. A method for detecting a novel coronavirus COVID-19 nucleic acid for non-disease diagnostic purposes, comprising the steps of:

s5, constructing the ssRNA nucleic acid probe of claim 2 or 3, wherein the activated Cas13a enzyme arbitrarily cleaves the ssRNA nucleic acid probe to generate a fluorescent signal, thereby rapidly and accurately identifying and detecting the novel coronavirus COVID-19 nucleic acid.

6. The method of claim 5, wherein the step S1 of preparing the viral protein and nucleic acid complex by human engineering comprises: cloning a target gene of the gene to a cell expression vector, transfecting cells after sequencing verification, then processing the cells to collect protein and nucleic acid compounds, removing genomic DNA residues by nuclease, extracting nucleic acid by a TRIZOL method, and calculating copy number to be used as a transcription-mediated amplification template.

7. The method of claim 5, wherein in step S2, 20 μ L of each component of the transcription-mediated amplification reaction system is gently vortexed and mixed, and then placed in a water bath at 42 ℃ for reaction for 15-25min to obtain the ssRNA transcript, wherein the transcription-mediated amplification reaction system comprises the following components at the following concentrations: 2 mu.L of positive strand RNA fragment to be amplified, 1-5U RNase inhibitor, 500-2000U T7 RNA polymerase, 2000-4000U M-MLV reverse transcriptase, 0.5-2 mu M upstream primer, 0.5-2 mu M downstream primer and TMA reaction buffer; the positive strand RNA fragment to be amplified is the nucleic acid extracted in step S1;

8. The method of claim 5, wherein in step S4, the components of the "ternary complex" system are mixed by gentle vortex, and then are subjected to heat preservation by a real-time fluorescence quantitative instrument at 37-40 ℃ for 10-25min, and then fluorescence is collected every 40-60S, and then collected for 40-60min in a circulating manner; the "ternary complex" system comprises the following components in the following concentrations: RNA fragment to be detected, 0.5-2 muM TMA upstream primer, 0.5-2 muM TMA downstream primer, 2000-4000U M-MLV reverse transcriptase, 500-2000U T7 RNA polymerase, 1-5U RNase inhibitor, 50-200nM cas13a protease, 50-400nM sgRNA, 0.25-1 muM RNA nucleic acid probe, 20-50mM Tris-HCl pH7.5@25 ℃, 20-40mM KCl, 1-5mM MgCl₂1-5mM rNTPs, 1-5mM dNTPs, 20-50% glycerol and 0.5-1mM DTT.