Primer set, double constant temperature color reagent kit and detection method for 2019 novel coronavirus lamp detection

Abstract

The application provides a primer set, a double constant temperature color developing kit and a detection method for 2019 novel coronavirus LAMP detection, and the double constant temperature color developing kit comprises outer primer pair 1, outer primer pair 2, inner primer pair 1, inner primer pair 2, loop primer pair 1 and loop primer pair 2. The 2019 novel coronavirus double constant temperature color developing screening detection kit provided by the application has the advantages of mature and stable technology, low testing cost, and is more suitable for the rapid screening and detection of community laboratories, enterprise laboratories, county health clinics and other primary laboratories, and has good market competitiveness and good industrialization prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to primer sets, dual isothermal colorimetric reagent kits, and detection methods for the LAMP detection of the 2019 novel coronavirus. Background Technology

[0002] Existing COVID-19 test kits have several drawbacks. PCR technology is simple, rapid, and highly sensitive, but it is labor-intensive, cumbersome, and has low throughput. Gene chips offer high throughput but are prone to false positives and are expensive. Real-time quantitative PCR (qPCR) technology is highly sensitive and reliable, and is a fully enclosed reaction; however, it requires expensive qPCR instruments, hindering its widespread adoption in grassroots testing departments and epidemic prevention institutions. Furthermore, ensuring high specificity requires fluorescently labeled probes, increasing testing costs. In short, these nucleic acid testing technologies all suffer from drawbacks such as reliance on expensive instruments, high testing costs, and laboratory limitations. Moreover, these molecular biology tests require high standards for quality control, operating environments, and personnel expertise, preventing widespread application at the grassroots level and failing to meet the need for rapid virus detection during an epidemic. Isothermal colorimetric amplification is simple and convenient to operate, requiring no highly skilled personnel; however, its complex primer design leads to false negatives or false positives, resulting in a shortage of isothermal colorimetric amplification reagents.

[0003] Currently, the quality of nucleic acid test kits varies greatly, and the stability and reliability of nucleic acid testing remain questionable. The detection rate is low, requiring many cases to be tested 2-3 times. Many patients have negative throat swabs but the virus is present in lung cell lavage fluid. The number of people waiting for nucleic acid testing far exceeds testing capacity, leading to unreliable results from hastily administered tests. Recent reports indicate that multiple institutions have successfully isolated and sequenced the entire genome of the 2019-nCoV novel coronavirus and conducted variant analysis. Compared to the 2019-nCoV reference genome NC 045512.2, many strains have undergone two important mutations in the ORF 1ab gene, suggesting that existing detection methods and targets need further updates and optimization based on viral mutations. It is necessary to continue research on novel coronavirus N gene detection methods, improve detection specificity and sensitivity, and develop rapid screening techniques suitable for on-site testing, providing convenient and efficient technical means for on-site detection. Summary of the Invention

[0004] To address the aforementioned problems in the prior art, this invention provides a primer set for the LAMP detection of the 2019 novel coronavirus. Another objective of this invention is to provide a dual isothermal colorimetric screening kit and its detection method for the LAMP detection of the 2019 novel coronavirus. The detection method of this invention does not require special instruments to obtain results, and is rapid and low-cost.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A set of primers for LAMP detection of the 2019 novel coronavirus, comprising outer primer pair 1, inner primer pair 1, and loop primer pair 1, wherein the sequences of outer primer pair 1 are shown in SEQ ID No. 1 and SEQ ID No. 2, the sequences of inner primer pair 1 are shown in SEQ ID No. 3 and SEQ ID No. 4, and the sequences of loop primer pair 1 are shown in SEQ ID No. 5 and SEQ ID No. 6.

[0007] A set of primers for LAMP detection of the 2019 novel coronavirus, comprising outer primer pair 2, inner primer pair 2, and loop primer pair 2, wherein the sequences of outer primer pair 2 are shown in SEQ ID No. 7 and SEQ ID No. 8, the sequences of inner primer pair 2 are shown in SEQ ID No. 9 and SEQ ID No. 10, and the sequences of loop primer pair 2 are shown in SEQ ID No. 11 and SEQ ID No. 12.

[0008] A primer set for LAMP detection of the 2019 novel coronavirus, comprising outer primer pair 1, outer primer pair 2, inner primer pair 1, inner primer pair 2, loop primer pair 1, and loop primer pair 2. The sequences of outer primer pair 1 are shown in SEQ ID No. 1 and SEQ ID No. 2, the sequences of outer primer pair 2 are shown in SEQ ID No. 7 and SEQ ID No. 8, the sequences of inner primer pair 1 are shown in SEQ ID No. 3 and SEQ ID No. 4, the sequences of inner primer pair 2 are shown in SEQ ID No. 9 and SEQ ID No. 10, the sequences of loop primer pair 1 are shown in SEQ ID No. 5 and SEQ ID No. 6, and the sequences of loop primer pair 2 are shown in SEQ ID No. 11 and SEQ ID No. 12.

[0009] The application of the primer set described above in the preparation of a LAMP detection kit for detecting the 2019 novel coronavirus.

[0010] A dual isothermal colorimetric screening kit for LAMP detection of the 2019 novel coronavirus, the kit comprising: the primer set as described above.

[0011] The dual isothermal colorimetric screening kit described above also includes strand displacement Bst DNA polymerase and reverse transcriptase.

[0012] The dual isothermal colorimetric screening kit described above also includes calcein, dNTPs, KCl, MgCl2, a positive control, and a negative control. The positive control is a plasmid containing the in vitro transcribed RNA sequence of the N gene, and the negative control is sterile water with nucleic acid removed.

[0013] A method for LAMP detection of the 2019 novel coronavirus, which is a non-diagnostic detection method, specifically includes the following steps:

[0014] (1) Extract RNA from the sample;

[0015] (2) The RNA extracted in step (1) is subjected to isothermal amplification; wherein, in the reaction system, the primer set used for LAMP detection of 2019 novel coronavirus as described above is used and the reaction is placed at a constant temperature of 63°C.

[0016] (3) After the constant temperature reaction has been carried out for 30 minutes, the reaction is removed and the results are observed;

[0017] (4) Result determination: If the sample is green, it is judged as positive; if it is light orange, it is judged as negative.

[0018] In the isothermal colorimetric method described above, preferably, in step (2), the isothermal amplification reaction system is as follows: the total reaction volume is 25 μL, wherein the reaction system contains N-1: reaction solution: 8 × 10 5 U / L Rnasin, 200mM dNTPs, 10mM Tris-HCl (pH=8.3), 20mM KCl, 3.5mM MgCl2, 0.8M betaine, inner primer pair 1 and inner primer pair 2 with a final concentration of 3.2μM, outer primer pair 1 and outer primer pair 1 with a final concentration of 0.4μM, and loop primer pair 1 and loop primer pair 2 with a final concentration of 1.6μM, totaling 21.3μL, calcein 1μL, Bst polymerase 1.5μL, AMV enzyme 0.2μL, and sample RNA template 2.0μL.

[0019] In the isothermal colorimetric method described above, preferably, nuclease-free water is set as a negative control, and a plasmid containing the sequence of in vitro transcribed RNA of the N gene is used as a positive control for detection.

[0020] The beneficial effects of this invention are as follows:

[0021] This invention provides a primer set for LAMP detection of the 2019 novel coronavirus, containing two sets of LAMP detection primers. These primers can be used separately for the detection of the 2019 novel coronavirus, or used in combination for more sensitive and accurate detection. This invention, through close analysis of the internationally published complete genome sequence of the novel coronavirus, including intraspecific conservation comparison, extraspecific homology comparison, and differential site analysis of the N gene, designed and screened four inner primers, four outer primers, and four loop primers as core technologies, enhancing the specificity and sensitivity of the isothermal colorimetric amplification screening detection method and kit. Compared with similar technologies at home and abroad, the results have significant cost and benefit advantages. The developed novel coronavirus N gene dual isothermal colorimetric screening detection kit has performance indicators equivalent to RT-PCR, and the detection time can be completed in 35-45 minutes, making it simple and fast to operate. This invention provides a novel coronavirus N gene dual isothermal colorimetric amplification primer set (inner and outer loops), along with a developed isothermal colorimetric screening method and kit. This addresses the issue of the lack of convenient isothermal colorimetric screening methods and kits for detecting the N gene, which are currently only available via isothermal amplification chip methods and real-time quantitative RT-PCR. It serves as a powerful supplement to existing methods and kits for detecting the novel coronavirus N gene. The novel coronavirus N gene dual isothermal colorimetric screening kit boasts advantages such as mature and stable technology, low testing cost, and suitability for grassroots implementation. It is particularly suitable for rapid screening in community laboratories, enterprise laboratories, and county health centers, demonstrating strong market competitiveness and promising industrialization prospects. Attached Figure Description

[0022] Figure 1 Differential sites compared to Bat SARS-like coronavirus;

[0023] Figure 2 Differential sites compared to SARS coronavirus;

[0024] Figure 3 These are the results of primer screening;

[0025] Figure 4 Sensitivity test amplification results. Detailed Implementation

[0026] The following examples are used to further illustrate the present invention, but should not be construed as limiting the invention. Modifications or substitutions made to the present invention without departing from its spirit and essence are all within the scope of the invention. The AMV reverse transcriptase BioLabs M0277, Bst polymerase BioLabs M0537L isothermal fluorescence amplification reaction solution used in the examples of this invention were purchased from Guangzhou Double Helix Technology Co., Ltd.; the isothermal water bath was purchased from Chunqiu Electronic Instruments Co., Ltd.; and the isothermometer was purchased from Tianjin Tiangen Biotech Co., Ltd. Reagents and instruments from other companies can also be used. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0027] Example 1: Screening of N gene dual isothermal chromogenic amplification primer set

[0028] 1.1 Sequence alignment of specifically amplified gene fragments

[0029] 1.1.1 Intraspecific conservation comparison

[0030] The novel coronavirus is an enveloped, single-stranded, positive-sense RNA virus with a genome length of approximately 30 kb. Four structural proteins play crucial roles in virion assembly. The S gene encodes a spike glycoprotein, which is used to enclose the viral capsid. The M gene encodes a membrane glycoprotein responsible for transmembrane transport of nutrients, budding and release of new viruses, and the formation of the viral envelope. The E gene is a short envelope glycoprotein encoding 75 amino acids that binds to the viral envelope. The N gene encodes a nucleocapsid protein, a structural protein involved in virion assembly, playing a key role in viral transcription and assembly efficiency.

[0031] The 26 2019-nCoV sequences downloaded from the National Science and Technology Resource Service System for Novel Coronavirus (http: / / nmdc.cn / # / nCoV) showed a homology of over 99.92% within the 2019-nCoV species after comparative analysis, with only a few base discrepancies at the ends.

[0032] 1.1.2 In vitro homology comparison

[0033] Preliminary comparison results show that it shares 91% homology with two bat SARS coronaviruses (bat-COVZC45 and bat-SL-COVZXC21) and approximately 88% homology with the SARS virus. After excluding other bat coronaviruses, it is found to share approximately 53% homology with the MERS coronavirus, with a conservation of approximately 63%.

[0034] 1.1.3 Differential site analysis

[0035] The novel coronavirus is an RNA virus, characterized by its instability and high mutation rate. Inconsistent bases are distributed throughout the entire genome, with approximately 1-2 inconsistent bases out of every dozen or so bases. Comparison with differentially expressed sites analysis of bat-COVZC45 and SARS MA15 yields the following results: Figure 1 , Figure 2 As shown.

[0036] 1.2 Primer design

[0037] Based on the results of the above comparison, the conserved region of the N gene was selected as the detection target. Four sets of isothermal chromogenic amplification primer pairs were designed using Primer Premier 5.0 software, labeled N1#Primer, N2#Primer, N3#Primer, and N4#Primer, respectively. Each primer set included two inner primers, two outer primers, and two loop primers, and was evaluated using Primer blast (http: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). A positive control of in vitro transcribed RNA from the novel coronavirus N gene was used for primer screening, and the chromogenic amplification reaction results and specificity of the four designed primer sets were analyzed.

[0038] 1.3 Specific amplification test of primer set

[0039] 1.3.1 Preparation of positive control

[0040] Based on the 2019-nCoV nucleic acid sequence published by the National Science and Technology Resource Service System for Novel Coronavirus (http: / / nmdc.cn / # / nCoV), positive control samples of in vitro transcribed RNA from the N gene plasmid were artificially synthesized, aliquoted, and stored at -20℃ for later use.

[0041] 1.3.2 Specific amplification assay

[0042] Nucleic acid or positive control plasmids of other common pathogens with the same infection site or similar infection symptoms were extracted, mainly including coronavirus 229E plasmid, coronavirus OC43 plasmid, coronavirus HKU1 plasmid, coronavirus NL63 plasmid, MERS coronavirus plasmid, influenza A virus RNA, influenza B virus RNA, parainfluenza virus plasmid, adenovirus plasmid, respiratory syncytial virus plasmid, rhinovirus plasmid, mycoplasma pneumoniae plasmid, chlamydia pneumoniae plasmid, streptococcus pneumoniae plasmid, Klebsiella pneumoniae nucleic acid, Legionella nucleic acid, Staphylococcus aureus nucleic acid, etc. for specific amplification tests.

[0043] 1.3.3 Results of specific amplification test of primer set

[0044] Primers were screened using a positive control of the novel coronavirus N gene in vitro transcribed RNA. The colorimetric amplification reaction results and specificity of the four designed primer sets were analyzed. Through screening of the four primer sets, the N1#Primer and N2#Primer primer sets showed earlier and more stable colorimetric development (e.g., ...). Figure 3 (As shown), primers N3#Primer and N4#Primer did not detect any signal. The initially selected N1#Primer + N2#Primer primer set was used in a combined tube experiment to establish a dual isothermal colorimetric detection method; finally, the N1#Primer + N2#Primer primer combination tube was determined as the inner and outer loop primer set for dual isothermal colorimetric amplification of the novel coronavirus N gene for subsequent experiments. The inner and outer loop primer set for dual isothermal colorimetric amplification of the 2019 novel coronavirus N gene includes the following primers:

[0045] (1) External primer pair 1:

[0046] SEQ ID NO.1: 5′-CCCCAAAATCAGCGAAATGC-3′

[0047] SEQ ID NO.2: 5′-CCACCACGAATTCGTCTGG-3′

[0048] (2) Inner primer pair 1:

[0049] SEQ ID NO.3: 5′-CGTTGTTTTGATCGCGCCCCTTTCATTACGTTTGGTGGACCCT-3′

[0050] SEQ ID NO.4: 5′-AATTCCCTCGAGGACAAGGCGTTTTAGCTCTTCGGTAGTAGCCA-3′

[0051] (3) Loop primer pair 1:

[0052] SEQ ID NO.5: 5′-TGGTTACTGCCAGTTGAATC-3′

[0053] SEQ ID NO. 6: 5'-TAACACCAATAGCAGTCCAGATG-3'.

[0054] (1) External primer pair 2:

[0055] SEQ ID NO.7: 5′-CACCCGCAATCCTGCTAAC-3′

[0056] SEQ ID NO.8: 5′-TTTGCTCTCAAGCTGGTTCA-3′

[0057] (2) Inner primer pair 2:

[0058] SEQ ID NO.9: 5′-CCTCTGCTCCCTTCTGCGTAGATTTAATGCTGCAATCGTGCTACA-3′

[0059] SEQ ID NO.10: 5′-AACTCCAGGCAGCAGTAGGGTTTGTCAAGCAGCAGCAAAGC-3′

[0060] (3) Loop primer pair 2:

[0061] SEQ ID NO.11: 5′-GCCTTTGGCAATGTTGTTCCTT-3′

[0062] SEQ ID NO. 12: 5'-CTCCTGCTAGAATGGCTGGC-3'.

[0063] Example 2: Optimization of the S-gene isothermal colorimetric amplification detection system

[0064] 2.1 Establishment of the isothermal colorimetric amplification reaction system

[0065] Using 10 5 U / L Bst enzyme, 4×10 6 U / L AMV enzyme, 8×10 5 A premixed reaction solution for isothermal colorimetric amplification was prepared using U / L RNasin, 200 mM dNTPs, 10 mM Tris-HCl (pH = 8.3), 20 mM KCl, and 3.5 mM MgCl2. The novel coronavirus N gene detection primers confirmed in Example 1 were used to prepare a dual isothermal colorimetric amplification detection reaction system for the novel coronavirus N gene. After mixing, the system was placed in an isothermal instrument for isothermal colorimetric amplification.

[0066] The reaction conditions are:

[0067] Constant temperature water bath: react at 63℃ for 30 minutes.

[0068] Or, use an isothermal amplification instrument: react at 63℃ for 30 minutes.

[0069] 2.2 Establishment and optimization results of the isothermal colorimetric amplification detection system

[0070] The test sample was 1 ng / μL of N gene in vitro transcribed RNA. The test system was determined to show a green color for positive samples and an orange-yellow color for negative samples within a reaction time of 30 minutes. The final reaction system is shown in Table 1.

[0071] Table 1. Isothermal Colorimetric Amplification Detection System

[0072]

[0073] The above-mentioned inner primer pairs 1 and 2 refer to the four inner primers each having a concentration of 3.2 μM, outer primer pairs 1 and 2 refer to the four outer primers each having a concentration of 0.4 μM, and loop primer pairs 1 and 2 refer to the four loop primers each having a concentration of 1.6 μM.

[0074] Example 3: Sensitivity Validation of the N Gene Dual Isothermal Chromogenic Amplification Screening Detection Method

[0075] Positive control samples of the N protein gene in vitro transcribed RNA plasmid were diluted 10-fold to obtain gradient concentrations of 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, and 1 fg / μL. The samples were then tested using the isothermal colorimetric amplification reaction system described in Example 2. The results showed that within 45 minutes, both parallel samples of the 10 fg amplification were stably detected, while only one parallel sample of the 1 fg amplification was detected, indicating a detection limit of 10 fg.

[0076] Example 4: Novel Coronavirus N Gene Dual Isothermal Colorimetric Screening Detection Kit and its Usage Method

[0077] The novel coronavirus S protein gene isothermal colorimetric screening detection kit prepared by this invention includes the sequence shown in the primer set in Example 1.

[0078] The kit can be configured to provide a single test kit quantity for convenient use, as shown in Table 1. The kit is packaged according to the required number of tests. Positive and negative controls may also be included. The positive control is N gene in vitro transcribed RNA, and the negative control is RNase-free dH2O. The kit is suitable for the following sample types: throat swabs, nasopharyngeal swabs, anal swabs, plasma, etc. Specimens should avoid repeated freeze-thaw cycles.

[0079] Required equipment and insulation devices: constant temperature water bath or isothermal detector; pipettes (range 0.1-200uL); sample rack and float (for water bath only); several latex or disposable gloves.

[0080] The specific steps for the testing are as follows:

[0081] RNA extraction from samples (or other extraction kits can be used).

[0082] (I) Sample RNA Extraction Steps

[0083] 1. Take the preservation solution of clinical samples such as pharyngeal swabs, nasopharyngeal swabs, anal swabs, and plasma into a 1.5mL centrifuge tube, add 200μL of solution No. 1 to each tube, add 3μL of carrier RNA (1μg / μL), vortex to mix for 15s, and let stand at room temperature for 3-5min.

[0084] 2. Add 20 μL of magnetic beads to the centrifuge tube (Note: Shake well before adding the magnetic beads), then add 150 μL of solution No. 2. Gently invert to mix, then place the tube on a magnetic rack. Once the magnetic beads have settled to the bottom of the tube, aspirate and discard the supernatant (Note: Do not aspirate the magnetic beads; the centrifuge tube should remain on the magnetic rack while aspirating the supernatant).

[0085] 3. Add 200 μL of solution No. 3 to the centrifuge tube, mix thoroughly by inverting, place it back on the magnetic rack, and wait for the magnetic beads to collect at the bottom of the tube before aspirating and discarding the supernatant.

[0086] 4. Repeat step 3.

[0087] 5. Open the centrifuge tube caps and place at 55°C for 4 minutes before removing them.

[0088] 6. Add 20 μL of solution #4, mix well with a pipette, and return to the magnetic rack. The supernatant is the extracted RNA.

[0089] (II) Dual-Isothermal Colorimetric Amplification Detection Procedure

[0090] (1) Take out the kit, thaw the reagents completely, centrifuge each component for 30s. BI and RI are liquids. After taking them out, place them on an ice box.

[0091] (2) Reagent preparation

[0092] If there are N samples to be tested, calculate the usage of each component according to the following formula (N+3 samples + 1 negative control + 1 positive control + 1 spare), for a total usage of:

[0093] N-1 21.3×(N+3)μL

[0094] B-1 1.5×(N+3)μL

[0095] R-1 0.2×(N+3)μL

[0096] Total reaction volume: 23 × (N + 3) μL

[0097] Place the reaction solution in a 0.6 mL or 1.5 mL centrifuge tube, vortex to mix, centrifuge for 30 s, and aliquot 23 μL into a 0.2 mL PCR tube.

[0098] (3) Add a template for the sample to be tested

[0099] Add 2 μL of template (RNA or DNA of the sample to be tested) to each of the 0.2 mL PCR tubes containing the reaction solution from step (2), centrifuge for 30 s, and proceed with the amplification reaction immediately.

[0100] (4) Amplification reaction

[0101] Isothermal or water bath conditions: Reaction at 63℃ for 30 min.

[0102] (5) Judgment of test results

[0103] For the detection system to be effective, all blank controls, negative controls, and positive controls must meet the following conditions: within a reaction time of 30 minutes, the positive control should show a green color, and the negative control should show an orange-yellow color.

[0104] Sample Result Interpretation: The sample test result should be compared with the positive and negative controls. If the sample reaction solution appears green, the test result is positive; if the sample reaction solution appears orange-yellow, the test result is negative. Observing the results against a black background is more conducive to the observation of experimental results. sequence list <110> Tianjin Normal University <120> Primer set, dual isothermal colorimetric reagent kit and detection method for LAMP detection of 2019 novel coronavirus. <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 ccccaaaatc agcgaaatgc 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <400> 2 ccaccacgaa ttcgtctgg 19 <210> 3 <211> 43 <212> DNA <213> Artificial Sequence <400> 3 cgttgttttg atcgcgcccc tttcattacg tttggtggac cct 43 <210> 4 <211> 44 <212> DNA <213> Artificial Sequence <400> 4 aattccctcg aggacaaggc gttttagctc ttcggtagta gcca 44 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 tggttactgc cagttgaatc 20 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <400> 6 taacaccaat agcagtccag atg 23 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <400> 7 cacccgcaat cctgctaac 19 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 tttgctctca agctggttca 20 <210> 9 <211> 45 <212> DNA <213> Artificial Sequence <400> 9 cctctgctcc cttctgcgta gatttaatgc tgcaatcgtg ctaca 45 <210> 10 <211> 41 <212> DNA <213> Artificial Sequence <400> 10 aactccaggc agcagtaggg tttgtcaagc agcagcaaag c 41 <210> 11 <211> twenty three <212> DNA <213> Artificial Sequence <400> 11 gccttttggc aatgttgttc ctt 23 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 ctcctgctag aatggctggc 20

Claims

1. A set of primers for LAMP detection of the 2019 novel coronavirus, characterized in that, It includes outer primer pair 1, outer primer pair 2, inner primer pair 1, inner primer pair 2, loop primer pair 1, and loop primer pair 2. The sequences of outer primer pair 1 are shown in SEQ ID No. 1 and SEQ ID No. 2, the sequences of outer primer pair 2 are shown in SEQ ID No. 7 and SEQ ID No. 8, the sequences of inner primer pair 1 are shown in SEQ ID No. 3 and SEQ ID No. 4, the sequences of inner primer pair 2 are shown in SEQ ID No. 9 and SEQ ID No. 10, the sequences of loop primer pair 1 are shown in SEQ ID No. 5 and SEQ ID No. 6, and the sequences of loop primer pair 2 are shown in SEQ ID No. 11 and SEQ ID No.

12.

2. The application of the primer set according to claim 1 in the preparation of a LAMP detection kit for detecting the 2019 novel coronavirus.

3. A dual isothermal colorimetric screening kit for LAMP detection of the 2019 novel coronavirus, characterized in that, The kit includes the primer set as described in claim 1.

4. The dual isothermal colorimetric screening kit according to claim 3, characterized in that, The kit also includes strand displacement Bst DNA polymerase and reverse transcriptase.

5. The dual isothermal colorimetric screening kit according to claim 3, characterized in that, The kit also includes calcein, dNTPs, KCl, MgCl2, a positive control, and a negative control. The positive control is a plasmid containing the in vitro transcribed RNA sequence of the N gene, and the negative control is sterile water with nucleic acid removed.

6. A method for LAMP detection of the 2019 novel coronavirus, characterized in that, This method is a non-diagnostic testing method, specifically including the following steps: (1) Extract RNA from the sample; (2) The RNA extracted in step (1) is subjected to isothermal amplification; wherein, in the reaction system, the primer set for LAMP detection of 2019 novel coronavirus as described in claim 1 is used and the reaction is carried out at a constant temperature of 63°C. (3) After the constant temperature reaction has been carried out for 30 minutes, the reaction is removed and the results are observed; (4) Result determination: If the sample is green, it is judged as positive; if it is light orange, it is judged as negative.

7. The method according to claim 6, characterized in that, In step (2), the isothermal amplification reaction system is as follows: the total reaction volume is 25 μL, wherein the reaction system contains N-1: reaction solution: 8×105U / L Rnasin, 200mM dNTPs, 10mM Tris-HCl, pH=8.3, 20mM KCl, 3.5mM MgCl2, 0.8M betaine, inner primer pair 1 and inner primer pair 2 with a final primer concentration of 3.2 μM, outer primer pair 1 and outer primer pair 1 with a final primer concentration of 0.4 μM, and loop primer pair 1 and loop primer pair 2 with a final primer concentration of 1.6 μM, totaling 21.3 μL, calcein 1 μL, Bst polymerase 1.5 μL, AMV enzyme 0.2 μL, and sample RNA template 2.0 μL.

8. The method according to claim 6, characterized in that, Meanwhile, nuclease-free water was set up as a negative control; a plasmid containing the sequence of in vitro transcribed RNA of the N gene was used as a positive control for detection.

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