CN116479170A - Novel coronavirus SARS-CoV-2 nucleic acid detection kit - Google Patents
Novel coronavirus SARS-CoV-2 nucleic acid detection kit Download PDFInfo
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Abstract
The invention discloses a novel coronavirus SARS-CoV-2 nucleic acid detection kit, belonging to the technical field of biological nucleic acid detection. The novel coronavirus SARS-CoV-2 nucleic acid detection primer set of the invention comprises at least one of a SARS-CoV-2 specific gene S primer set or a SARS-CoV-2 specific gene N primer set. The invention designs 2 groups of specific primers aiming at N genes and S genes based on RT-LAMP technology, and utilizes Bst DNA polymerase with strand displacement activity and heat-resistant RT enzyme to rapidly detect SARS-CoV-2, the sensitivity reaches 10-20cp/mL, the invention can carry out high-efficiency specific strand displacement amplification reaction under the constant temperature condition (60-65 ℃), overcomes the defect that qRT-PCR reaction needs to obtain a single-stranded template through a heat denaturation process, and has low equipment requirement, and can be detected in situ in real time.
Description
Technical Field
The invention belongs to the technical field of biological nucleic acid detection, and in particular relates to a novel coronavirus SARS-CoV-2 nucleic acid detection kit.
Background
For the nucleic acid detection of novel coronaviruses SARS-CoV-2, the real-time fluorescence reverse transcription polymerase chain reaction (qRT-PCR) detection method is still the preferred method for SARS-CoV-2 nucleic acid detection, but qRT-qPCR requires multiple steps, high cost reagents and expensive laboratory instruments, and is not suitable for point-of-care nucleic acid detection (POCT) or home self-test.
The loop-mediated isothermal amplification (LAMP) technology can realize on-site rapid detection without depending on special instruments, the reaction temperature is 60-65 ℃, the reaction can be carried out only by a simple heater such as a water bath kettle and the like, and the result can be visually judged. The existing commercial SARS-CoV-2 nucleic acid RT-LAMP (reverse transcription loop-mediated isothermal amplification) kit is mainly used for detecting most of extracted nucleic acids, is not lower in use cost than qRT-PCR, and is low in specificity and easy to generate a false positive result in non-specific amplification. Many studies to solve the problem that non-specific amplification will bind to CRISPR technology on RT-LAMP basis, which undoubtedly increases the detection cost even further. Nucleic acid extraction reagents are expensive and require specialized personnel and extraction equipment, while increasing detection time. In order to solve the problem, a lysis reagent matched with RT-LAMP is invented, and the sample is directly amplified after being treated in a lysis solution for 10min or heat treatment for 5min at 95 ℃ aiming at extracting-free one or more samples such as a throat swab, saliva or nose swab, and the operation is still relatively complicated. Therefore, the invention discloses a nucleic acid extraction-isothermal amplification-hybridization integrated system such as a microfluidic cartridge, a microfluidic chip and the like, the relative price is relatively high, and the monitoring, prevention and control requirements of a basic layer on the pandemic epidemic situation can not be met.
One difficulty with loop-mediated isothermal amplification (LAMP) technology is primer design, which requires the use of 6 pairs of special primers to identify 6 segments of the target gene, and the longer sequences of the inner primers FIP and BIP, and the addition of loop primers LF and LB increases the amplification rate, so that primer mixtures tend to form primer dimers in long-term storage. The optimal reaction temperature of LAMP is 60-65 ℃ and 95 ℃ is not existed, so that the amplification sensitivity is reduced, and the sensitivity of the existing commercial SARS-CoV-2 nucleic acid RT-LAMP kit is 40 copies/reaction-80 copies/reaction, which is far lower than qRT-PCR; the detection time is 30-50min, and although the detection time is slightly shortened compared with that of the conventional qRT-PCR, the rapid qRT-PCR reagent can also reach the detection time.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a novel coronavirus SARS-CoV-2 nucleic acid detection kit which can rapidly detect the novel coronavirus SARS-CoV-2 and can judge the result visually so as to reduce the detection time and cost.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a novel coronavirus SARS-CoV-2 nucleic acid detection primer set comprises at least one of a SARS-CoV-2 specific gene S primer set or a SARS-CoV-2 specific gene N primer set. The invention designs 2 groups of specific primers aiming at the N gene and the S gene, has high specificity, can detect 5 new coronasubtypes such as alpha, beta, delta, omicron, gamma of novel coronavirus, and can not leak detection on variant strains such as BA.1, BA.2, BA.3, BA.4, BA.5 and the like; the invention optimizes and screens the primer design, and finally optimizes the primer group with high amplification efficiency and high sensitivity, thereby realizing the completion of the reaction within 20 min.
As a preferred embodiment of the novel coronavirus SARS-CoV-2 nucleic acid detection primer set of the present invention, the SARS-CoV-2 specific gene S primer set comprises outer primers S-F3 and S-B3, inner primers S-FIP and S-BIP, and loop primers S-LF and S-LB; the nucleotide sequence of the S-F3 is shown as SEQ ID NO. 1; the nucleotide sequence of the S-B3 is shown as SEQ ID NO. 2; the nucleotide sequence of the S-FIP is shown as SEQ ID NO. 3; the nucleotide sequence of the S-BIP is shown as SEQ ID NO. 4; the nucleotide sequence of the S-LF is shown as SEQ ID NO. 5; the nucleotide sequence of the S-LB is shown as SEQ ID NO. 6.
As a preferred embodiment of the novel coronavirus SARS-CoV-2 nucleic acid detection primer set of the present invention, the SARS-CoV-2 specific gene N primer set comprises outer primers N-F3 and N-B3, inner primers N-FIP and N-BIP, and loop primers N-LF and N-LB; the nucleotide sequence of the N-F3 is shown as SEQ ID NO. 7; the nucleotide sequence of the N-B3 is shown as SEQ ID NO. 8; the nucleotide sequence of the N-FIP is shown as SEQ ID NO. 9; the nucleotide sequence of the N-BIP is shown as SEQ ID NO. 10; the nucleotide sequence of the N-LF is shown as SEQ ID NO. 11; the nucleotide sequence of the N-LB is shown as SEQ ID NO. 12.
The invention also provides the application of the novel coronavirus SARS-CoV-2 nucleic acid detection primer group in preparing a novel coronavirus SARS-CoV-2 nucleic acid detection product.
The invention also provides a novel coronavirus SARS-CoV-2 nucleic acid detection kit, which comprises a freeze-drying reaction premix, a sampling tube, a quantitative dropper and a sample collector.
As a preferred embodiment of the novel coronavirus SARS-CoV-2 nucleic acid detection kit of the present invention, the lyophilization reaction premix comprises a 5 xRT-LAMP reaction solution, a primer set and a lyophilization complex-protecting agent. The freeze-dried premix of the invention combines the required reaction components according to the corresponding proportion, and is dissolved and used after being re-dissolved by the sampling liquid and the sample mixed liquid, thus avoiding repeated sample adding, being more convenient and accurate to use and being more suitable for the field detection of a base layer.
As a preferred embodiment of the novel coronavirus SARS-CoV-2 nucleic acid detection kit of the present invention, the 5 xRT-LAMP reaction solution comprises the following components in concentration: 1-5mM Tris-HCl (pH 8.8), 10-35mM KCl,10-25mM MgSO 4 ，15-40mM(NH 4 ) 2 SO 4 0.05-1.0% Tween 20, 12-40mM dNTPs,1-8U/ul Bst DNA polymerase, 10-50U/ul RT enzyme, 1-5U/ul RNase inhibitor, 50-150mM pH indicator. More preferably, the indicator is cresol red. The pH indicator is added into the freeze-dried premix liquid, the result can be interpreted by naked eye color change, the negative and positive color change difference is obvious, the interpretation is visual and convenient, and the uncapping identification of the amplified product after the reaction is avoidedThe secondary pollution of aerosol caused by the substances reduces the exposure risk in the detection process.
As a preferred embodiment of the novel coronavirus SARS-CoV-2 nucleic acid detection kit, the freeze-dried composite protectant comprises the following components in parts by weight: 3-5 parts of trehalose, 2-4 parts of dimethyl sulfoxide, 2-5 parts of mannose and 0.3-2 parts of vitamin D. According to the invention, the composite freeze-drying protective agent is added into the reaction premix, and the reaction premix is prepared into freeze-drying for storage and transportation, so that the stability of the reagent is greatly improved, the non-specific amplification is reduced, the normal-temperature transportation is convenient, and the transportation cost is saved. More preferably, the lyoprotectant further comprises 15-55 μmol/ml KOH.
As a preferred embodiment of the novel coronavirus SARS-CoV-2 nucleic acid detection kit of the present invention, the sampling tube comprises a nucleic acid releasing agent, a nucleic acid preserving fluid and a nucleic acid buffer.
As a preferred embodiment of the novel coronavirus SARS-CoV-2 nucleic acid detection kit of the present invention, the sample comprises a nasal swab sample, a pharyngeal swab sample or a saliva sample. The nucleic acid releasing agent can directly amplify samples such as throat swab, nose swab and saliva by hands-free taking, and can be used for on-site instant detection or self-detection by matching with a small portable amplification instrument.
The invention has the beneficial effects that: the invention provides a novel coronavirus SARS-CoV-2 nucleic acid detection kit, which is based on RT-LAMP technology, designs 2 groups of specific primers for N gene and S gene, and utilizes Bst DNA polymerase with strand displacement activity and heat-resistant RT enzyme to rapidly detect SARS-CoV-2, the sensitivity reaches 10-20cp/mL, and can carry out high-efficiency specific strand displacement amplification reaction under the constant temperature condition (60-65 ℃), thereby overcoming the defects that qRT-PCR reaction needs to obtain a single-stranded template through a thermal denaturation process, and the like, and simultaneously has low requirement on equipment and can be detected in situ immediately; the kit is simple and convenient to operate, has low technical quality requirements on detection personnel, and can realize low-cost rapid screening on large samples; the detection of the kit provided by the invention only needs 10-20 min, and the result can be judged by naked eyes, so that the kit is more visual and clear; according to the kit disclosed by the invention, the composite freeze-drying protective agent is added into the reaction premix, and the reaction premix is prepared into freeze-drying for storage and transportation, so that the stability of the reagent is greatly improved, the non-specific amplification is reduced, the normal-temperature transportation is convenient, and the transportation cost is saved.
Drawings
FIG. 1 is an amplification curve of the novel coronavirus SARS-CoV-2 nucleic acid detection kit of example 2 for different target reaction concentrations;
FIG. 2 is a graph showing the amplification color reaction of the novel coronavirus SARS-CoV-2 nucleic acid detection kit of example 2 for different target reaction concentrations;
FIG. 3 is a diagram showing the results of specific detection of the novel coronavirus SARS-CoV-2 nucleic acid detection kit of example 3;
FIG. 4 is a diagram showing the results of the universal assay of the novel coronavirus SARS-CoV-2 nucleic acid assay kit of example 4;
FIG. 5 is a graph showing the results of the novel coronavirus SARS-CoV-2 nucleic acid assay kit of example 5 for different sample types;
FIG. 6 is a graph of the results of the detection of novel coronavirus SARS-CoV-2 by the lyophilized and non-lyophilized reaction premixes.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.
Example 1 preparation of novel coronavirus SARS-CoV-2 nucleic acid detection kit
1. Designing RT-LAMP primer, synthesizing and preparing primer group solution: a novel coronavirus gene sequence database (https:// bigd.big.ac.cn/ncov) of the national genome science data center is registered. The genome sequences of novel coronaviruses, other coronaviruses, and other pneumoviruses (influenza virus, respiratory syncytial virus, rhinovirus, adenovirus, parainfluenza virus, enterovirus, herpes simplex virus, human metapneumovirus, reovirus, measles virus, cytomegalovirus, and the like) were searched and downloaded, and analyzed by using bioinformatics software such as CLUSTAL, DNASTAR, BIOEDIT. Analysis shows that the genome sequence among the isolated strains of the novel coronavirus SARS-CoV-2 which is popular recently has no obvious variation, so that the genome sequence NC_045512 of the representative strain of the novel coronavirus SARS-CoV-2 is selected as a template to design a primer. The genomic sequences of SARS-CoV-2 and coronaviruses such as SARS-Cov, MERS-Cov, HKU1, OC43, NL63 and 229E were then aligned and screened for regions of the genomic sequences specific for SARS-CoV-2 that are suitable for the design of RT-LAMP primers. Meanwhile, the template region sequences screened by BLAST on-line tool analysis can be specifically aligned to the gene sequences of SARS-CoV-2, but not to other coronavirus gene sequences, and also to the gene sequences of influenza virus, respiratory syncytial virus, rhinovirus, adenovirus, parainfluenza virus, enterovirus, herpes simplex virus, human metapneumovirus, reovirus, measles virus, cytomegalovirus and the like. The quality of RT-LAMP primer determines the specificity and sensitivity of RT-LAMP detection method, when designing primer, the formation of primer dimer should be avoided as much as possible, and 6 regions identified by primer are specific regions matched with S gene or N gene of SARS-CoV-2, so that all S gene or N gene of SARS-CoV-2 can be identified, and thus all novel coronavirus SARS-CoV-2 can be detected. The invention designs RT-LAMP specific amplification primers by using S gene and N gene of novel coronavirus SARS-CoV-2 as templates and on-line software Primer Explore V5. The S gene RT-LAMP primer group comprises 6 primers: the outer primers S-F3 and S-B3, the inner primers S-FIP and S-BIP, the loop primers S-LF and S-LB, and the N gene RT-LAMP primer set comprises 6 primers: outer primers N-F3 and N-B3, inner primers N-FIP and N-BIP, and loop primers N-LF and N-LB.
The gene sequences of the primers are as follows:
S-F3：5’-TGTGTTAATCTTACAACCAGAA-3’(SEQ ID NO:1)；
S-B3：5’-GGAAGCAAAATAAACACCATCA-3’(SEQ ID NO:2)；
S-FIP:5’-AACTGAGGATCTGAAAACTTTGTCA-TCAATTACCCCCTGCAT AC-3’(SEQ ID NO:3)；
S-BIP:5’-TTACATTCAACTCAGGACTTGTTCT-TTATCAAACCTCTTAGTA CCATTG-3’(SEQ ID NO:4)；
S-LF：5’-GGGTAATAAACACCACGTGTGA-3’(SEQ ID NO:5)；
S-LB：5’-TTTCCAATGTTACTTGGTTCCATGC-3’(SEQ ID NO:6)；
N-F3：5’-AACACAAGCTTTCGGCAG-3’(SEQ ID NO:7)；
N-B3：5’-GAAATTTGGATCTTTGTCATCC-3’(SEQ ID NO:8)；
N-FIP：5’-CGCATTGGCATGGAAGTCACTTTGATGGCACCTGTGTAG-3’
(SEQ ID NO:9)；
N-BIP:5’-TGCGGCCAATGTTTGTAATCAGCCAAGGAAATTTTGGGGAC-3’(SEQ ID NO:10)；
N-LF：5’-TTCCTTGTCTGATTAGTTC-3’(SEQ ID NO:11)；
N-LB：5’-ACCTTCGGGAACGTGGTT-3’(SEQ ID NO:12)。
primers were synthesized by the division of biological engineering (Shanghai) and purified by HPLC. The primers F3, B3 and FIP, BIP, LF, LB were prepared as 25 Xmixed aqueous solutions with final molar concentrations of 1:1:8:8:2:2, respectively, using DEPC water to prepare aqueous solutions with a certain molar concentration.
In the detection, 1ul of primer group solution is added into each 25ul of reaction system, so that the mole numbers of the primers in each reaction system are respectively as follows: primers F3 and B3 were 0.2uM, FIP and BIP were 1.6uM, LF and LB were 0.4uM.
2. Preparing a 5 xRT-LAMP reaction solution: the 5 xRT-LAMP reaction solution contains the following components in concentration: 20mM Tris-HCl (pH 8.8), 20mM KCl,16mM MgSO 4 ，20mM(NH 4 ) 2 SO 4 0.2% Tween 20, 16mM dNTPs,1.28U/ul Bst DNA polymerase, 16U/ul RT enzyme, 1.6U/ul RNase inhibitor, 100mM pH indicator cresol red.
In the detection, 5. Mu.L of 5 xRT-LAMP reaction solution was added to each reaction system (25 ul) so that each component and its final concentration were 4mM Tris-HCl (pH 8.8), 10mM KCl,8mM MgSO 4 ，10mM(NH 4 ) 2 SO 4 0.1% tween-20,1.6mM dNTPs,8U Bst DNA polymerase, 20U RT enzyme, 20U rnase inhibitor, 50mM cresol red.
3. And (3) assembling a kit: the detection kit is composed of a freeze-drying reaction premix 1 tube/person, a sampling 1 tube/person, a quantitative dropper 1, a nasal swab 1 person, a specification and a portable small-sized amplification instrument.
Example 2
This example measures the detection sensitivity of the novel coronavirus SARS-CoV-2 nucleic acid detection kit of example 1, and the specific experimental method is as follows: 5. Mu.L of 5 xLAMP reaction solution, 1ul of primer set aqueous solution, 1ul of positive template and the total volume of sterilized DEPC water to 25ul were added to each RT-LAMP reaction system (25 ul). Each 25ul of the RT-LAMP reaction system had a final concentration of 4mM Tris-HCl (pH 8.8), 10mM KCl,8mM MgSO 4 ，10mM(NH 4 ) 2 SO 4 0.1% tween-20,1.6mM dNTPs,8UBst DNA polymerase, 20U RT enzyme, 2.5U rnase inhibitor, 50mM cresol red, moles of each primer: primers F3 and B3 were each 0.2uM, and primers FIP, BIP were each 1.6uM, LF and LB were each 0.4uM. The test was run with negative and blank controls.
The novel coronavirus nucleic acid standard substance (high concentration) GBW (E) 091089 is added into the RT-LAMP reaction system, so that the target reaction concentration is respectively 200cp/T, 120cp/T, 80cp/T, 40cp/T, 10cp/T and 5cp/T, the reaction time is 45min, the reaction temperature is 65 ℃, and the amplification curve is drawn through fluorescence monitoring, so that the sensitivity is measured.
The experimental results are shown in figures 1-2, and the detection sensitivity of the kit provided by the invention reaches 10cp/T.
Example 3
The detection specificity of the novel coronavirus SARS-CoV-2 nucleic acid detection kit of example 1 is detected in this example, and the specific experimental method is as follows: 5. Mu.L of 5 xLAMP reaction solution, 1ul of primer set aqueous solution, 1ul of positive template and the total volume of sterilized DEPC water to 25ul were added to each RT-LAMP reaction system (25 ul). Each 25ul of the RT-LAMP reaction system had a final concentration of 4mM Tris-HCl (pH 8.8), 10mM KCl,8mM MgSO 4 ，10mM(NH 4 ) 2 SO 4 0.1% tween-20,1.6mM dNTPs,8UBst DNA polymerase, 20U RT enzyme, 2.5U rnase inhibitor, 50mM cresol red, moles of each primer: primers F3 and B3 were each 0.2uM, and primers FIP, BIP were each 1.6uM, LF and LB were each 0.4uM.
And respectively adding SARS-CoV-2, SARS-CoV, MERS-CoV, influenza A, influenza B and Rhinovalve standard substances, performing specific cross test, and visually judging the detection result by visual observation of the color under natural light at 65 ℃ for 20min, and comparing the influence of different reaction temperatures on RT-LAMP detection.
Observing the color of the reaction liquid under natural light, and judging that the sample to be detected contains novel coronavirus SARS-CoV-2 if the color of the reaction liquid changes to orange; if the color of the reaction solution is still the original mauve, the sample to be tested is judged to contain no novel coronavirus SARS-CoV-2. As shown in FIG. 3, the primer of the present invention has high specificity and no cross amplification phenomenon, and the primer combination of the present invention only amplifies N gene and S gene of novel coronavirus, but not coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV).
Example 4
This example demonstrates the versatility of the novel coronavirus SARS-CoV-2 nucleic acid detection kit of example 1, and the specific experimental procedure is as follows: 5. Mu.L of 5 xLAMP reaction solution, 1ul of primer set aqueous solution, 1ul of positive template and the total volume of sterilized DEPC water to 25ul were added to each RT-LAMP reaction system (25 ul). Each 25ul of the RT-LAMP reaction system had a final concentration of 4mM Tris-HCl (pH 8.8), 10mM KCl,8mM MgSO 4 ，10mM(NH 4 ) 2 SO 4 0.1% tween-20,1.6mM dNTPs,8UBst DNA polymerase, 20U RT enzyme, 2.5U rnase inhibitor, 50mM cresol red, moles of each primer: primers F3 and B3 were each 0.2uM, and primers FIP, BIP were each 1.6uM, LF and LB were each 0.4uM. The test was set as a negative control.
And (3) respectively adding alpha, beta, delta, gamma, omicron (including BA.1, BA.2, BA.3, BA.4 and BA.5) strain standard substances, performing a universality test, performing a reaction time of 20min at 65 ℃, visually inspecting the color under natural light, visually judging the detection result, and comparing the detection conditions of different mutant strains.
Observing the color of the reaction liquid under natural light, and judging that the sample to be detected contains novel coronavirus SARS-CoV-2 if the color of the reaction liquid changes to orange; if the color of the reaction solution is still the original mauve, the sample to be tested is judged to be free of the novel coronavirus SARS-CoV-2, and the kit has very good universality through testing. As shown in FIG. 4, the primer set of the invention has strong inclusion and universality, and can detect the new crown variant strain which is popular at present: alpha, beta, delta, gamma and ommicon (omacron), and ba.1, ba.2, ba.3, ba.4, ba.5.
Example 5
The detection of the novel coronavirus SARS-CoV-2 nucleic acid visual detection kit of example 1 in this example is different in sample type interference resistance, and the specific experimental method is as follows: 5. Mu.L of 5 xLAMP reaction solution, 1ul of primer group aqueous solution and 1ul of positive template are added into each RT-LAMP reaction system (25 ul), and the negative human nasal swab sample, pharyngeal swab sample and saliva sample are used for supplementing the volume to 25ul. Each 25ul of the RT-LAMP reaction system had a final concentration of 4mM Tris-HCl (pH 8.8), 10mM KCl,8mM MgSO 4 ，10mM(NH 4 ) 2 SO 4 0.1% tween-20,1.6mM dNTPs,8U Bst DNA polymerase, 20U RT enzyme, 2.5U rnase inhibitor, 50mM cresol red, moles of each primer: primers F3 and B3 were each 0.2uM, and primers FIP, BIP were each 1.6uM, LF and LB were each 0.4uM. The test was set as a negative control.
And respectively adding equivalent negative humanized nose swab samples, throat swab samples, saliva samples and virus standard substances into an amplification system, performing amplification reaction, visually judging the detection result by visual observation of the color under natural light at 65 ℃ for 20min, and comparing the influence of different samples on RT-LAMP detection.
As shown in FIG. 5, the amplification system of the kit has strong anti-interference capability, can perform direct amplification reaction on samples such as nasal swab, pharyngeal swab, saliva and the like, and has detection rate of more than 95%.
Example 6
This example provides a novel coronavirus SARS-CoV-2 nucleic acid detection kit, which differs from the kit of example 1 only in that: the reaction premix of the kit does not contain a freeze-drying composite protective agent, does not carry out freeze-drying treatment, and is liquid premix.
The above-mentioned kit and the kit of example 1 were stored for one week, and then subjected to novel coronavirus SARS-CoV-2 nucleic acid detection, and the results are shown in FIG. 6. After 1 week of storage in the form of a liquid premix, some non-specific amplification occurred and false positives occurred (as shown in fig. 6A, the first 4 negative samples changed from 2 to yellow positive); however, the reagent premix was prepared in lyophilized form and stored for 3 months without non-specificity and without false positives (as shown in fig. 6B, the first 4 negative samples were all normal red negative).
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A novel coronavirus SARS-CoV-2 nucleic acid detection primer group is characterized by comprising at least one of a SARS-CoV-2 specific gene S primer group or a SARS-CoV-2 specific gene N primer group.
2. The novel coronavirus SARS-CoV-2 nucleic acid detection primer set of claim 1, wherein said SARS-CoV-2 specific gene S primer set comprises outer primers S-F3 and S-B3, inner primers S-FIP and S-BIP, and loop primers S-LF and S-LB; the nucleotide sequence of the S-F3 is shown in SEQ ID NO. 1; the nucleotide sequence of the S-B3 is shown as SEQ ID NO. 2; the nucleotide sequence of the S-FIP is shown as SEQ ID NO. 3; the nucleotide sequence of the S-BIP is shown as SEQ ID NO. 4; the nucleotide sequence of the S-LF is shown as SEQ ID NO. 5; the nucleotide sequence of the S-LB is shown as SEQ ID NO. 6.
3. The novel coronavirus SARS-CoV-2 nucleic acid detection primer set of claim 1, wherein said SARS-CoV-2 specific gene N primer set comprises outer primers N-F3 and N-B3, inner primers N-FIP and N-BIP, and loop primers N-LF and N-LB; the nucleotide sequence of the N-F3 is shown as SEQ ID NO. 7; the nucleotide sequence of the N-B3 is shown as SEQ ID NO. 8; the nucleotide sequence of the N-FIP is shown as SEQ ID NO. 9; the nucleotide sequence of the N-BIP is shown as SEQ ID NO. 10; the nucleotide sequence of the N-LF is shown as SEQ ID NO. 11; the nucleotide sequence of the N-LB is shown as SEQ ID NO. 12.
4. Use of the novel coronavirus SARS-CoV-2 nucleic acid detection primer set as claimed in any one of claims 1 to 3 for preparing a novel coronavirus SARS-CoV-2 nucleic acid detection product.
5. A novel coronavirus SARS-CoV-2 nucleic acid detection kit is characterized by comprising a freeze-drying reaction premix, a sampling tube, a quantitative dropper and a sample collector.
6. The novel coronavirus SARS-CoV-2 nucleic acid detection kit as claimed in claim 5, wherein the lyophilization reaction premix comprises a 5 xRT-LAMP reaction solution, a primer set and a lyophilization complex protectant.
7. The novel coronavirus SARS-CoV-2 nucleic acid detection kit of claim 6, wherein the 5 xrt-LAMP reaction solution comprises the following concentration components: 1-5mM Tris-HCl (pH 8.8), 10-35mM KCl,10-25mM MgSO 4 ，15-40mM(NH 4 ) 2 SO 4 0.05-1.0% Tween 20, 12-40mM dNTPs,1-8U/ul Bst DNA polymerase, 10-50U/ul RT enzyme, 1-5U/ul RNase inhibitor, 50-150mM pH indicator.
8. The novel coronavirus SARS-CoV-2 nucleic acid detection kit of claim 6, wherein the freeze-dried composite protectant comprises the following components in parts by weight: 3-5 parts of trehalose, 2-4 parts of dimethyl sulfoxide, 2-5 parts of mannose and 0.3-2 parts of vitamin D.
9. The novel coronavirus SARS-CoV-2 nucleic acid detection kit as claimed in claim 5, wherein the sampling tube comprises a nucleic acid releasing agent, a nucleic acid preserving fluid, a nucleic acid buffer.
10. The novel coronavirus SARS-CoV-2 nucleic acid detection kit of claim 5, wherein the sample comprises a nasal swab sample, a pharyngeal swab sample, or a saliva sample.
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