A probe set, a probe set for detecting multiple nucleic acid targets of a novel coronavirus, a kit and a detection method thereof

Abstract

This invention discloses a probe set, a probe set for detecting multiple nucleic acid targets of the novel coronavirus, a kit, and a detection method. Based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology, combined with endonuclease and a mediator-universal probe (reporter probe) signal reporting mode, this invention achieves specific analysis of dual target genes of the novel coronavirus within 90 minutes in a fluorescent probe system designed independently of the target sequence. The system achieves a sensitivity of 100 copies / reaction for both genes, exhibits good specificity, and shows no cross-signaling. This method is helpful for accurately and rapidly screening patients infected with the novel coronavirus and is of great significance for the diagnosis, treatment, and prevention and control of COVID-19.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and mainly relates to a probe set, a probe set for detecting multiple nucleic acid targets of the novel coronavirus, and its reagent kit and detection method. Background Technology

[0002] Currently, the diagnosis of novel coronavirus infection mainly relies on real-time quantitative RT-PCR (RT-qPCR) for viral nucleic acid load detection or next-generation sequencing for viral nucleic acid sequence analysis. However, RT-qPCR viral nucleic acid detection requires the test DNA template, specific primers, fluorescent dye, and PCR amplification buffer to undergo a certain number of amplification cycles under PCR amplification conditions to reach a set fluorescence signal threshold. Next-generation sequencing requires the addition of dNTPs during the generation of new DNA complementary strands to catalyze the substrate through an enzymatic cascade reaction to excite fluorescence, or the direct addition of fluorescently labeled dNTPs or semi-degenerate primers to release fluorescence signals during the synthesis or ligation of complementary strands. By capturing the light signal and converting it into a sequencing peak, the complementary strand sequence information can be obtained. Therefore, RT-qPCR and next-generation sequencing have a series of problems, including long detection times and cycles, high environmental requirements, high technical expertise requirements, high hardware requirements, and high costs.

[0003] In recent years, isothermal amplification technology has been widely used in the detection of pathogen nucleic acids. Among them, reverse transcription / loop-mediated isothermal amplification (RT- / LAMP) is the most prominent due to its low environmental requirements, lack of need for expensive thermal cycling equipment, short reaction time, high sensitivity and specificity, and low cost. LAMP uses at least six target primer regions (four primers) to efficiently amplify the target at a constant temperature (60-65℃), achieving a detection sensitivity of up to 100 copies / reaction, with a reaction time of approximately 20-45 minutes. Furthermore, the Bst DNA polymerase used in LAMP has good tolerance to reaction inhibitors, allowing for amplification and detection through simple sample processing—such as heating. Currently, there are numerous reports on the application of LAMP methods in the detection of SARS-CoV-2, but most are single-target detection modes. However, SARS-CoV-2 belongs to the single-stranded RNA coronavirus genus and exhibits characteristics such as high mutation rates (e.g., B1.1.7, B1.351), making single-target detection prone to false negative results.

[0004] Currently, methods for detecting SARS-CoV-2 virus using loop-mediated isothermal amplification include LAMP primer sets described in Chinese patents: CN202010150878.9, CN202011429713.1, CN202011167529.4, CN202010312576.7, CN202011045783.7, and CN202010150527.8. These known primer sets only target the six target primer regions of the SARS-CoV-2 virus's target genes. The LAMP method uses four to six primers, offering high sensitivity and a short reaction time (30-60 minutes). Clinical use requires no special equipment (a real-time turbidimeter is recommended during reagent kit development). The procedure is simple (for both DNA and RNA, the detection steps involve mixing the reaction solution, enzyme, and template in a PCR tube, incubating at approximately 63°C for 30-60 minutes, and visually observing the results). Besides turbidimetric detection, calcein can be added to the reaction system. After the LAMP reaction, Mn... 2+ It binds to the reaction product pyrophosphate, releasing calcein and thus releasing the quenched state, exhibiting yellow-green fluorescence. However, due to the addition of Mn... 2+ This reduced the detection sensitivity by an order of magnitude; some researchers added the nucleic acid dye SYBR Green I to the reaction solution, but due to the limitations of SYBR Green... I has a certain inhibitory effect on the LAMP reaction. Among the improved methods for LAMP detection of other target genes, Ball et al. proposed a simple RT-LAMP endpoint detection technique, which enabled the detection of West Nile virus and Chikungunya virus RNA. This method first labels the circular primer or inner primer with a fluorescent dye, and then hybridizes it with a short chain labeled with a quencher group. Thus, when LAMP amplification is not performed in the entire reaction system, there is no strong fluorescence signal. When the target template is present, LAMP amplification begins, and the primer labeled with the fluorescent dye binds to a specific site of the target template, thereby freeing the short chain of the quencher group in the solution. As a result, the reaction system exhibits a strong fluorescence signal. However, this method requires the addition of the cationic polymer PEI after the reaction is completed following LAMP amplification. PEI forms a precipitate with the LAMP product, removing all amplification products containing the circular primer or inner primer to prevent complementary hybridization with the short chain labeled with the quencher group. The short chain of the quencher group is then separated from the LAMP product with the fluorescent dye, and the fluorescence intensity is then detected. Therefore, the current methods for detecting SARS-CoV-2 virus based on LAMP are limited in their widespread use because real-time turbidity analyzers are expensive and unsuitable for on-site detection, and the quantitative fluorescence method that has been tried suffers from reduced sensitivity or slower reaction due to the addition of indicators, and specific fluorescent probes cannot detect in real time.

[0005] Therefore, there is an urgent need for a simple, sensitive, specific, low-cost, universal, and rapid nucleic acid detection method for the novel coronavirus that can detect multiple targets. Summary of the Invention

[0006] The problem to be solved by this invention is to improve the existing real-time fluorescence quantitative detection method for SARS-CoV-2 virus based on LAMP, so as to detect LAMP reaction products in real time without reducing sensitivity or inhibiting LAMP reaction.

[0007] The concept of this invention is based on LAMP, which efficiently amplifies the targets of two target genes of the SARS-CoV-2 virus using at least six target primer regions (six primers) at a constant temperature (60-65°C). The product formed after amplification is a stem-loop DNA strand. Simultaneously, a mediator probe and a fluorescent reporter probe are introduced during loop-mediated isothermal amplification. Signals are generated by activating the mediator and interacting with a specific reporter probe. The mediator probe includes a target-specific sequence. The mediator sequence does not bind to any region of the stem-loop DNA strand. When the target-specific sequence pairs complementaryly with a specific region of the stem-loop DNA strand, the RNA bases on the target-specific sequence (which are cleavage sites) are cleaved by the endonuclease RNase HII. Under the action of Bst DNA polymerase, the continuously generated mediator sequence with 3' hydroxyl free activity binds to the specific reporter probe, displacing the corresponding fluorescent channel signal. Without template DNA or RNA, LAMP amplification of the target gene cannot be achieved, thus preventing the generation of stem-loop DNA strands and the formation of double strands that are complementary to the target-specific sequence. Consequently, the mediator sequence cannot be cleaved, and fluorescent channel signals (positive signals) cannot be formed.

[0008] The technical solution of the present invention is: a probe set comprising a reporter probe and at least one mediator probe, wherein the mediator probe comprises a mediator sequence and a target-specific sequence from the 5' to 3' direction, the target-specific sequence comprising a sequence complementary to a partial control sequence of the nucleic acid target sequence of the novel coronavirus, the partial control sequence of the nucleic acid target sequence being: a sequence amplified along the loop after the nucleic acid target sequence of the novel coronavirus is complementary to a circular primer or an inner primer in a loop-mediated isothermal amplification reaction, the mediator sequence comprising a sequence not complementary to the nucleic acid target sequence or the control sequence, one base of the target-specific sequence being modified into an RNA base and the remainder being DNA bases, the RNA base being located at the 2nd or 3rd base from the 5' end of the target-specific sequence;

[0009] The reporter probe comprises, from 3' to 5', a capture sequence complementary to the mediator sequence or a portion thereof, and a template sequence, wherein the RNA bases and the target-specific sequences at the RNA base ends are not complementary to the reporter probe. The reporter probe is labeled with a reporter group at its 5' end and a quencher group at its 3' end. The signal emitted by the reporter probe when hybridizing with its complementary sequence is different from the signal emitted when it does not hybridize with its complementary sequence. The reporter group and the quencher group are spaced 10-80 nt or longer apart.

[0010] Advantageously, after the circular primer or inner primer pairs complementaryly with the nucleic acid target sequence of the novel coronavirus, a stem-loop DNA strand is amplified. The target-specific sequence specifically binds to the stem-loop DNA strand to form a double strand. Furthermore, one base in the target-specific sequence is modified into an RNA base. The mediator sequence contains a sequence that is not complementary to the nucleic acid target sequence or control sequence. Additionally, the RNA base is 2-3 bases away from the 5' end of the target-specific sequence and 1 base away from the 3' end of the mediator sequence. These are necessary conditions for the mediator sequence to be cleaved by RNase HII. After the mediator sequence is complementary to the reporter probe, the signal emitted by the reporter group is positively correlated with the copy number of the stem-loop DNA strand.

[0011] Furthermore, the nucleic acid target sequence of the novel coronavirus is selected from the conserved gene sequence of the novel coronavirus.

[0012] Furthermore, the nucleic acid target sequence of the novel coronavirus is selected from the conserved gene sequences of the SARS-CoV-2 virus: the N gene and the E gene.

[0013] Specifically, the probe group is selected from the following probe groups:

[0014] The first probe set is a loop primer targeting the E gene of SARS-CoV-2, which includes a loop mediator probe E-LMP with a nucleotide sequence as shown in SEQ ID No. 7, and one of two reporter probes UP1 / UP2 as shown in SEQ ID No. 8 and 18, respectively. The 5' end of the reporter probe is labeled with a fluorescent group, and the 3' end is labeled with a quencher group. The 16th base from the 5' to the 3' direction of the nucleotide sequence shown in SEQ ID No. 7 is modified to be an RNA base, and the rest are DNA bases.

[0015] The second probe set, targeting the E gene of SARS-CoV-2, includes an inner primer containing an inner mediator probe E-LMP with a nucleotide sequence as shown in SEQ ID No. 9, and one of two reporter probes UP1 / UP2 as shown in SEQ ID No. 8 and 18, respectively. The reporter probe is labeled with a fluorescent group at its 5' end and a quencher group at its 3' end. The 16th base from the 5' to the 3' direction of the nucleotide sequence shown in SEQ ID No. 9 is modified to be an RNA base, and the rest are DNA bases.

[0016] The third probe set consists of a loop primer targeting the N gene of SARS-CoV-2, comprising a loop mediator probe N-LMP with a nucleotide sequence as shown in SEQ ID No. 17, and one of two reporter probes UP1 / UP2 as shown in SEQ ID No. 8 and 18, respectively. The reporter probe is labeled with a fluorescent group at its 5' end and a quencher group at its 3' end. The 17th base from the 5' to the 3' direction of the nucleotide sequence shown in SEQ ID No. 17 is modified to be an RNA base, and the rest are DNA bases.

[0017] Specifically, the fluorescent groups at the 5' ends of the two reporter probes shown in SEQ ID No. 8 and 18 can be set to different fluorescent groups, and the template sequences of the two reporter probes shown in SEQ ID No. 8 and 18 are allowed to be different.

[0018] This invention also provides a probe set for detecting multiple nucleic acid targets of the novel coronavirus.

[0019] This includes primer sets for detecting the N gene of SARS-CoV-2, and / or primer sets for detecting the E gene of SARS-CoV-2.

[0020] The primer set for detecting the E gene of SARS-CoV-2 includes: the first probe set and the second probe set as described in claim 4, and the outer primer N-WF / N-WR of the E gene, the inner primer N-FIP / N-BIP of the E gene, and the circular primer N-LF / N-LB of the E gene.

[0021] The primer set for detecting the N gene of SARS-CoV-2 includes: the third probe set as described in claim 4, and the outer primer N-WF / N-WR, the inner primer N-FIP / N-BIP, and the circular primer N-LF / N-LB.

[0022] Furthermore, the outer primers for the N gene: Primer N-WF: nucleotide sequence as shown in SEQ ID No. 11; Primer N-WR: nucleotide sequence as shown in SEQ ID No. 12;

[0023] N gene inner primers: Primer N-FIP: nucleotide sequence as shown in SEQ ID No. 13; Primer N-BIP: nucleotide sequence as shown in SEQ ID No. 14;

[0024] N gene circular primers: Primer N-LF: nucleotide sequence as shown in SEQ ID No. 15; Primer N-LB: nucleotide sequence as shown in SEQ ID No. 16.

[0025] Furthermore, the outer primers for the E gene are as follows: Primer E-WF: nucleotide sequence as shown in SEQ ID No. 1; Primer E-WR: nucleotide sequence as shown in SEQ ID No. 2;

[0026] E gene inner primers: Primer E-FIP: nucleotide sequence as shown in SEQ ID No. 3; Primer E-BIP: nucleotide sequence as shown in SEQ ID No. 4;

[0027] E gene circular primers: Primer E-LF: nucleotide sequence as shown in SEQ ID No. 5; Primer E-LB: nucleotide sequence as shown in SEQ ID No. 6.

[0028] The present invention also provides a kit for detecting multiple nucleic acid targets of the novel coronavirus, comprising a solution containing the probe set in the primer set described above, wherein the primer set solution is a solution in which each primer or probe in the primer set is diluted to 0.05~1.6 μmol / L.

[0029] The above kit also includes 6 mmol MgSO4, 1.4 mmol dNTP (A / G / C / T), 0.32 U / μL BstDNA polymerase, 0.5 μL reverse transcriptase, 0.04 U / μL RNase HII, 5 μL template gene, and 1×Isothermal Amplification Buffer. The total volume of the detection system of the kit is 25 μL, and the remaining volume is made up to 25 μL with nuclease-free water.

[0030] Furthermore, the kit for detecting multiple nucleic acid targets of the novel coronavirus includes a solution containing a primer set for detecting the N gene of SARS-CoV-2 from the primer set mentioned above. The primer set solution consists of 0.2 μmol N-WF, 0.2 μmol N-WR, 1.6 μmol N-FIP, 1.6 μmol N-BIP, 0.8 μmol N-LF, 0.4 μmol N-LB, 0.4 μmol N-LMP, 0.2 μmol UP1 or UP2, and also includes 6 mmol MgSO4, 1.4 mmol dNTP (A / G / C / T), 0.32 U / μL Bst DNA polymerase, 0.5 μL reverse transcriptase, 0.04 U / μL RNase HII, 5 μL template gene, and 1×Isothermal Amplification Buffer. The total detection volume of the kit is 25 μL, and the remaining volume is made up to 25 μL with nuclease-free water.

[0031] Furthermore, the kit for detecting multiple nucleic acid targets of the novel coronavirus includes a solution containing a primer set for detecting the E gene of SARS-CoV-2 from the primer set mentioned above. The primer set solution consists of 0.2 μmol E-WF, 0.2 μmol E-WR, 1.6 μmol E-FIP, 0.8 μmol E-BIP, 0.8 μmol E-LF, 0.8 μmol E-LB, 0.8 μmol E-LMP, 0.2 μmol UP1 or UP2, and also includes 6 mmol MgSO4, 1.4 mmol dNTP (A / G / C / T), 0.32 U / μL LBst DNA polymerase, 0.5 μL reverse transcriptase, 0.04 U / μL RNase HII, 5 μL template gene, and 1×Isothermal Amplification Buffer. The total detection volume of the kit is 25 μL, and the remaining volume is made up to 25 μL with nuclease-free water.

[0032] Furthermore, the kit for detecting multiple nucleic acid targets of the novel coronavirus includes a solution containing a primer set for detecting the N gene of the novel coronavirus SAR-CoV-2 and a primer set for detecting the E gene of the novel coronavirus SAR-CoV-2.

[0033] The primer set for detecting the N gene of SARS-CoV-2 is composed of: 0.05 μmol N-WF, 0.05 μmol N-WR, 0.4 μmol N-FIP, 0.4 μmol N-BIP, 0.2 μmol N-LF, 0.1 μmol N-LB, 0.1 μmol N-LMP, and 0.2 μmol UP1 or UP2.

[0034] The primer set solution for detecting the E gene of SARS-CoV-2 is: 0.2 μmol E-WF, 0.2 μmol E-WR, 1.6 μmol E-FIP, 1.6 μmol E-BIP, 0.8 μmol E-LF, 0.4 μmol E-LB, 0.4 μmol E-LMP, and 0.2 μmol UP1 or UP2;

[0035] The kit also includes 6 mmol MgSO4, 1.4 mmol dNTPs (A / G / C / T), 0.32 U / μL Bst DNA polymerase, 0.5 μL reverse transcriptase, 0.04 U / μL RNase HII, 40 mmol guanidine hydrochloride, 5 μL template gene, and 1×Isothermal Amplification Buffer. The total volume of the kit's detection system is 25 μL, with the remaining volume brought to 25 μL with nuclease-free water.

[0036] This invention also provides a method for detecting multiple nucleic acid targets of the novel coronavirus, comprising the following steps:

[0037] S1. Sampling;

[0038] S2. Using the above-mentioned kit and the obtained sample, prepare the LAMP reaction system, place it in the reaction tube, mix and centrifuge;

[0039] S3. Place the reaction tube on a fluorescence spectrometer with a constant temperature function and react at 60~65℃. Collect fluorescence every 1 minute for 60~90 cycles.

[0040] S4. Read the Ct value. The criteria for judging the test results are as follows: if the Ct value of a sample is ≤60, the sample is judged to be positive.

[0041] If 60 < Ct value ≤ 75, and the amplification curve is a logarithmic amplification curve, the sample is judged as a suspected positive sample; otherwise, the sample is judged as negative.

[0042] For suspected positive samples, retesting is performed. If the Ct value of the retested sample is ≤60, the suspected positive sample is judged to be positive; otherwise, the sample is judged to be negative.

[0043] If a sample has no Ct value or a Ct value > 75, the sample is considered negative.

[0044] Furthermore, in step S3, if the kit in step S2 contains only primer sets for detecting the N or E gene, the amplification is performed at 65°C; if the kit in step S2 contains primer sets for detecting both the N and E genes, the amplification is performed at 60°C.

[0045] The present invention has the following beneficial effects:

[0046] The detection principle of the probe set of this invention is disclosed for the first time, and it is very different from the primers used in the existing RT-LAMP detection method in terms of detection principle.

[0047] The probe set includes six primers designed for at least six target primer regions of the N target gene and E target gene of SARS-CoV-2 virus, respectively. The six primers can be complementary to specific regions of the N target gene and E target gene, and then extend to form complementary sequences of the target genes.

[0048] It also includes designing different types of mediator probes for the inner primers and loop primers among the six primers. The mediator probes include mediator sequences and target-specific sequences. The target-specific sequences can pair complementaryly with specific sites of the complementary sequences of the target gene to form a double strand with a specific binding sequence containing an endonuclease cleavage site.

[0049] The two sets of LAMP primers, different types of mediator probes and a universal reporter probe, template DNA or RNA, reaction buffer, Bst DNA polymerase, and RNase HII are mixed and the target gene is efficiently amplified at a constant temperature (60-65℃). After amplification, a stem-loop DNA strand is formed. The target-specific sequence of the mediator probe binds to a specific site on the stem-loop DNA strand. The second or third base at the 5' end of the target-specific sequence is modified to be an RNA base, and the rest are DNA bases (which are cleavage sites). The mediator sequence does not bind to any region of the stem-loop DNA strand, so it stands up. Under the action of RNase HII, the mediator sequence is cleaved. Under the action of Bst DNA polymerase, the continuously generated mediator sequence with 3' hydroxyl free activity binds to the specific reporter probe, displacing the corresponding fluorescent channel signal. With each amplification cycle, the fluorescence signal, like the mediator sequence, undergoes a synchronous exponential growth process. The signal intensity corresponds to the copy number of the stem-loop DNA strand, and the fluorescence signal intensity can be read by a photomultiplier tube. However, in the absence of template DNA or RNA, LAMP amplification of the target gene cannot be achieved, thus preventing the generation of the stem-loop DNA strand and the restriction enzyme sites on the mediator probe, and consequently, the generation of a fluorescent channel signal (positive signal).

[0050] By combining endonuclease and mediator probe-universal probe (reporter probe) signal reporting mode, this invention achieves dual-target gene specificity analysis of the novel coronavirus within 90 minutes using a fluorescent probe system designed independently of the target sequence. The outer, inner, and circular primers for detecting the N and E genes of the novel coronavirus are not limited to the sequences provided in this invention. The N and E genes of the novel coronavirus are conserved gene sequences, and different types of novel coronaviruses show high homology in their N and E genes. The probe set provided in this invention can also be complementary to some control sequences of the N and E genes of different types of novel coronaviruses. The kit composed of the probe set designed in this invention can rapidly and accurately distinguish the novel coronavirus from the three most prevalent respiratory viruses each spring and winter. The reaction conditions are mild, the isothermal conditions are simple, and the detection equipment is simple, requiring only an isothermal water bath and a fluorescence instrument. Sensitive and rapid detection can be achieved in a single tube. The 5' ends of the reporter probe and mediator probe can be used in other different mixed primer sets. Therefore, the detection method of this invention is suitable for on-site detection, with high sensitivity, fast reaction, and real-time detection capabilities. Attached Figure Description

[0051] Figure 1 This is a schematic diagram of the principle of the universal real-time fluorescence quantitative detection method for nucleic acid targets based on LAMP of the present invention;

[0052] Figure 2 This is a comparison of the effectiveness of different probe types in the universal SARS-CoV-2 RT-LAMP E gene detection system. Figure 2 Pos indicates a positive experiment, and Neg indicates a negative experiment, meaning no probe was added.

[0053] Figure 3 This refers to the sensitivity of the universal SARS-CoV-2 RT-LAMP dual-target gene detection system when detecting plasmids carrying the N gene.

[0054] Figure 4 This refers to the sensitivity of the universal SARS-CoV-2 RT-LAMP dual-target gene detection system when detecting N gene RNA.

[0055] Figure 5 This refers to the sensitivity of the universal SARS-CoV-2 RT-LAMP dual-target gene detection system when detecting E gene RNA.

[0056] Figure 6 This refers to the specificity of the universal SARS-CoV-2 RT-LAMP dual-target gene detection system when using a reporter probe with a FAM fluorescent group;

[0057] Figure 7 This refers to the specificity of the universal SARS-CoV-2 RT-LAMP dual-target gene detection system when using a reporter probe with a HEX fluorescent group. Detailed Implementation

[0058] The technical solution of the present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. First, the reagents and terms designed in the present invention are explained as follows:

[0059] RNase HII is a ribonuclease that cleaves the 5' end of double-stranded DNA, producing a 5' phosphate terminus and a 3' hydroxyl terminus. RNase HII can also perform multiple cleavages on the RNA portion of the Okazaki fragment.

[0060] Bst DNA polymerase has 5′→3′ polymerase activity and double-strand-specific 5′→3′ exonuclease activity, but lacks 3′→5′ exonuclease activity.

[0061] Similar to SARS-CoV and MERS-CoV viruses, the genome of SARS-CoV-2 (2019-nCoV) is also divided into non-structural genes and structural genes. The non-structural genes, which comprise about two-thirds of the total genome length of SARS-CoV-2, contain two protein-coding sequences called ORF1a and ORF1b. The subsequent structural gene regions encode structural proteins such as the S protein, orf3a protein, E protein, M protein, orf6 protein, orf7a protein, orf8 protein, and N protein.

[0062] The novel coronavirus E gene expresses the E protein, which contains a hydrophobic domain and a transmembrane α-helical domain. It is a component of the viral envelope and participates in the assembly and release of viral particles. The E protein sequence of 2019-nCoV has a homology of up to 95% with that of SARS-CoV and is a conserved gene sequence. The novel coronavirus N gene expresses the N protein, which has a high degree of conserved sequence and binds to the RNA in the viral particles. It has a homology of up to 90% with the N protein of 2019-nCoV. In addition, the mutation frequency of the N protein is relatively low, and the different types are relatively similar [1], so it is often used as an antigen for immunological detection.

[0063] The open reading frame 1ab (ORF1ab) of the novel coronavirus is also a conserved gene sequence and can be used as a detection target.

[0064] [1] Ruan YJ, WeiCL, Ee AL, et al Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection[J]. Lancet, 2003, 361(9371):1779 - 1785.

[0065] The reaction principle of the LAMP-based universal real-time fluorescence quantitative detection method for nucleic acid targets of this invention is described in [the following text is incomplete and requires further context]. Figure 1 Six primers were designed for at least six target primer regions of the N and E target genes of the SARS-CoV-2 virus. The six primers can be complementary to specific regions of the N and E target genes and then extended to form complementary sequences of the target genes.

[0066] Different types of mediator probes were designed for the inner and loop primers of the six primers. The mediator probes include mediator sequences and target-specific sequences. The target-specific sequences can pair complementaryly with specific sites of the complementary sequences of the target gene to form a double strand with a specific binding sequence containing an endonuclease cleavage site.

[0067] The two sets of LAMP primers, different types of mediator probes and a universal reporter probe, template DNA or RNA, reaction buffer, Bst DNA polymerase, and RNase HII are mixed and the target gene is efficiently amplified at a constant temperature (60-65℃). After amplification, a stem-loop DNA strand is formed. The target-specific sequence of the mediator probe binds to a specific site on the stem-loop DNA strand. The second or third base at the 5' end of the target-specific sequence is modified to be an RNA base, and the rest are DNA bases (which are cleavage sites). The mediator sequence does not bind to any region of the stem-loop DNA strand, so it stands up. Under the action of RNase HII, the mediator sequence is cleaved. Under the action of Bst DNA polymerase, the continuously generated mediator sequence with 3' hydroxyl free activity binds to the specific reporter probe, displacing the corresponding fluorescent channel signal. After each amplification cycle, the fluorescence signal, like the mediator sequence, undergoes a synchronous exponential growth process. The signal intensity corresponds to the copy number of the DNA strand in the stem-loop structure, and the fluorescence signal intensity can be read out by a photomultiplier tube.

[0068] Without template DNA or RNA, LAMP amplification of the target gene cannot be achieved, which means that the stem-loop DNA strand and the restriction enzyme sites on the mediator probe cannot be generated, and therefore the fluorescent channel signal (positive signal) cannot be generated.

[0069] This invention therefore provides a method for sensitive and rapid detection of multiple target genes in a single tube. Since the circular mediator probe that specifically binds to the circular primer, the inner mediator probe that specifically binds to the inner primer, and the reporter probe are applicable in different multi-gene primer sets, this method is also known as a universal multiplex target gene detection method. That is, the 5' ends of the reporter probe and mediator probe can be used in other different mixed primer sets.

[0070] The SARS-CoV-2 genome (GenBank: MN908947.3) was downloaded, and specific N and E fragment sequences were obtained. RT-LAMP primer sets were designed based on these sequences.

[0071] Example 1

[0072] The specific implementation process of a typical embodiment of the present invention is as follows:

[0073] RT-LAMP primers were designed for six regions of the specific E fragment, including two outer primers E-WF / E-WR for the F3 / B3 region, two inner primers E-FIP / E-BIP, loop primers E-Loop-F / E-Loop-B (E-LF / E-LB) for the F2 and F1C regions and for the B2 and B1C regions, loop mediator probes Loop MP were designed for the amplification sequence of the loop primers E-LF / E-LB, and a reporter probe was designed for the sequence of the loop mediator probe Loop MP.

[0074] Example 2

[0075] The specific implementation process of a typical embodiment of the present invention is as follows:

[0076] RT-LAMP primers were designed for six regions of the specific E fragment, including two outer primers E-WF / E-WR for the F3 / B3 region, two inner primers E-FIP / E-BIP, and loop primers E-Loop-F / E-Loop-B (E-LF / E-LB) designed between the F2 and F1C regions and between the B2 and B1C regions.

[0077] Example 3

[0078] The specific implementation process of a typical embodiment of the present invention is as follows:

[0079] RT-LAMP primers were designed for six regions of the specific E fragment, including two outer primers E-WF / E-WR for the F3 / B3 region, two inner primers E-FIP / E-BIP, loop primers E-Loop-F / E-Loop-B (E-LF / E-LB) for the F2 and F1C regions and the B2 and B1C regions, inner mediator probes Inner MP were designed for the amplification sequence of the inner primers E-FIP / E-BIP, and a reporter probe was designed for the sequence of the inner mediator probe Inner MP.

[0080] Example 4

[0081] The specific implementation process of a typical embodiment of the present invention is as follows:

[0082] RT-LAMP primers were designed for six regions of the specific E fragment, including two outer primers E-WF / E-WR for the F3 / B3 region, two inner primers E-FIP / E-BIP, and loop primers E-Loop-F / E-Loop-B (E-LF / E-LB) designed between the F2 and F1C regions and between the B2 and B1C regions.

[0083] Example 5: Comparison of detection efficiency of different probe types in a universal SARS-CoV-2 RT-LAMP dual-target gene detection system.

[0084] To investigate the detection efficiency of the different types of loop and inner mediator probes provided in Examples 1-4, and the RT-LAMP primer sets designed for six regions of the specific E fragment, in a universal SARS-CoV-2-LAMP detection system, a certain concentration of SARS-CoV-2 RNA standard (Yisheng Biotechnology, catalog number: 11900ES03) was selected, i.e., 10... 4 The copy number per microliter was used as the system reaction template to investigate the amplification efficiency (Ct value) of different types of mediator probes in the universal SARS-CoV-2-LAMP E gene detection system. Specifically, the total volume of the universal SARS-CoV-2-LAMP E gene detection system is 25 μL. The specific components of the loop mediator probe system include: 1×Isothermal Amplification Buffer, 6 mM MgSO4, 1.4 mM dNTPs (A / G / C / T), 0.2 μM E-WF, 0.2 μM E-WR, 1.6 μM E-FIP, 1.6 μM E-BIP, 0.8 μM E-LF, 0.4 μM E-LB, 0.4 μM E-LMP, 0.2 μM UP2, 0.32 U / μL Bst 2.0 WarmStart® DNA Polymerase, 0.5 μL WarmStart RTx Reverse Transcriptase, 0.04 U / μL LRNase HII, 5 μL genomic DNA, and the remainder is made up to 25 μL with nuclease-free water. The system was reacted at 65°C on a fluorescence spectrometer with a temperature control function, and fluorescence was collected every 1 minute for 60-90 cycles. UP2 is a universal reporter probe: a TaqMan fluorescent probe, and U / μL indicates the number of drug units per microliter of drug.

[0085] The primer sequences for the E gene in Examples 1-4 are shown in Table 1 below:

[0086] Table 1

[0087]

[0088] The primer sequences used in the LAMP reaction system for detecting the E gene using a circular probe are shown in Table 2 below:

[0089] Table 2

[0090]

[0091] The total volume of the universal SARS-CoV-2-LAMP E gene detection system is 25 μL. The specific components of the inner mediator probe system include: 1×Isothermal Amplification Buffer, 6 mM MgSO4, 1.4 mM dNTP (A / G / C / T), 0.2 μM E-WF, 0.2 μM E-WR, 0.8 μM E-LF, 0.8 μM E-LB, 1.6 μM E-FIP, 0.8 μM E-BIP, 0.8 μM E-IMP, 0.2 μM UP2, 0.32 U / μL Bst 2.0 WarmStart® DNA Polymerase, 0.5 μL WarmStart RTx Reverse Transcriptase, 0.04 U / μL RNase HII, 5 μL genomic DNA, and the remainder is made up to 25 μL with nuclease-free water. The system was reacted at 65°C on a fluorescence spectrometer with a temperature control function, and fluorescence was collected every 1 minute for 60-90 cycles. UP2 is a universal reporter probe: a TaqMan fluorescent probe, and U / μL indicates the number of drug units per microliter of drug.

[0092] The primer sequences used in the LAMP reaction system for detecting the E gene using an internal vector probe are shown in Table 3 below:

[0093] Table 3

[0094]

[0095] Test results as follows Figure 2 As shown, the lowest detectable template copy concentration of qPCR was observed and compared when four mediator probes were used with primers for the E gene. It was found that when using four mediator probes for real-time PCR to detect the same concentration of SARS-CoV-2 RNA standard, the detection efficiency of the Loop MP probe with primers for the E gene was better than that of the InnerMP probe with primers for the E gene.

[0096] Example 6: Sensitivity Evaluation of a Universal SARS-CoV-2 RT-LAMP Dual-Target Gene Detection System

[0097] The specific implementation process of a typical embodiment of the present invention is as follows:

[0098] RT-LAMP primers were designed for six regions of the specific N fragment, including two outer primers (N-WF / N-WR) targeting the F3 / B3 region, two inner primers (N-FIP / N-BIP), and loop primers (N-Loop-F / N-Loop-B, N-LF / N-LB) designed between the F2 and F1C regions and between the B2 and B1C regions. The N gene sequence is shown in SEQ ID No. 10. An inner mediator probe (Loop MP-Pos) was designed based on the amplification sequence of the loop primers N-Loop-F / N-Loop-B. The plasmid carrying the N gene (pUC57 vector) was serially diluted, and the amplification efficiency (Ct value) of different serially diluted loop mediator probes for a universal SARS-CoV-2-LAMP N gene detection system was investigated. Specifically, the total volume of the universal SARS-CoV-2-LAMP N gene detection system is 25 μL. The specific components of the loop mediator probe system include: 1×IsothermalAmplification Buffer, 6 mM MgSO4, 1.4 mM dNTP (A / G / C / T), 0.2 μM N-WF, 0.2 μM N-WR, 1.6 μM N-FIP, 1.6 μM N-BIP, 0.8 μM N-LF, 0.4 μM N-LB, 0.4 μM N-LMP, 0.2 μM UP1, 0.32 U / μL Bst 2.0 WarmStart® DNA Polymerase, 0.5 μL WarmStart RTx Reverse Transcriptase, 0.04 U / μL RNase HII, 5 μL genomic DNA, and the remainder is made up to 25 μL with nuclease-free water. The system was reacted at 65°C on a fluorescence spectrometer with a thermostat, and fluorescence was collected every 1 minute for 60-90 cycles. UP1 was the reporter probe: a TaqMan fluorescent probe, and U / μL indicates the number of drug units per microliter. The primer sequences for the N gene are shown in Table 4 below.

[0099] Table 4

[0100]

[0101] like Figure 3 When the plasmid carrying the N gene is serially diluted to 10-1 6 10 5 10 4 10 3 10 2 5×10 1The copy number per microliter was used, with nuclease-free water as a negative control (Neg). At least three replicates were performed, and the Ct value was recorded. The judgment criteria for the test results are as follows: A sample Ct value ≤ 60 is considered positive; 60 < Ct value ≤ 75, if the amplification curve is a logarithmic amplification curve, is considered a suspected positive sample; otherwise, the sample is considered negative. Suspected positive samples were retested. If the retested sample Ct value ≤ 60, the suspected positive sample was considered positive; otherwise, the sample was considered negative; no Ct value or a Ct value > 75 indicates a negative sample.

[0102] The results showed that, after observing and comparing the detection sensitivity of five dilutions of qPCR, the detection limit of the N gene plasmid by the RT-LAMP method was 50 copies / reaction.

[0103] To investigate the sensitivity of RNA standards in a universal SARS-CoV-2 RT-LAMP dual-target gene detection system, a certain concentration of SARS-CoV-2 RNA standards (Yisheng Biotechnology, catalog number: 11900ES03) was selected, i.e., 10... 5 10 4 10 3 10 2 10 1 The copy number / µL was used as the reaction template, and a series of 10-fold serial dilutions were performed. Nuclease-free water was used as a negative control. At least three replicates were used for each assay. The reaction system for detecting N gene RNA is shown in Table 4, and the reaction system for detecting E gene RNA is shown in group 1 of Table 2. The results showed that... Figure 4 and Figure 5 The limit of detection for both N gene RNA and E gene RNA in the universal SARS-CoV-2 RT-LAMP dual-target gene detection system is 100 copies / reaction.

[0104] Example 7 Specificity Investigation of a Universal SARS-CoV-2 RT-LAMP Dual-Target Gene Detection System

[0105] To investigate the specificity of the universal SARS-CoV-2 RT-LAMP dual-target gene detection system, human respiratory syncytial virus (RSV), influenza A virus (InfA), and influenza B virus (InfB) were selected as targets for specificity evaluation. SARS-CoV-2 RNA standard was used as a positive control (Pos), and nuclease-free water as a negative control (Neg). At least three replicates were performed. The reaction system for detecting N gene RNA is shown in Table 4, and the reaction system for detecting E gene RNA is shown in group 1 of Table 2. The results showed that... Figure 6 and Figure 7The universal SARS-CoV-2 RT-LAMP dual-target gene detection system showed no cross-signals in different subjects, indicating good system specificity.

[0106] The RT-LAMP detection method of this invention is convenient for clinical application, that is, it can quickly and accurately distinguish it from the top three respiratory viruses that are prevalent every spring and winter.

[0107] The general SARS-CoV-2 nucleic acid target detection method of the present invention is based on the principle that, under the synergistic action of the endonuclease RNase HII and the DNA polymerase Bst DNA, in the presence of the target, the activation of the general mediator is achieved and it interacts with a specific reporter probe to generate a signal.

[0108] Example 8

[0109] The total volume of the universal SARS-CoV-2 RT-LAMP dual target gene detection system is 25 μL, and the components include: 1×Isothermal Amplification Buffer, 6 mM MgSO4, and 1.4 mM dNTPs (A / G / C / T);

[0110] 0.05 μM N-WF(SEQ ID No.13), 0.05 μM N-WR(SEQ ID No.14), 0.4 μM N-FIP(SEQ ID No.15), 0.4 μM N-BIP(SEQ ID No.16), 0.2 μM N-LF(SEQ ID No.17), 0.1 μM N-LB(SEQ ID No.18), 0.1 μM N-LMP (SEQ ID No. 19), 0.2 μM UP1 (SEQ ID No. 20);

[0111] 0.2 μM E-WF (SEQ ID No.1), 0.2 μM E-WR (SEQ ID No.2), 1.6 μM E-FIP (SEQ ID No.3), 1.6 μM E-BIP (SEQ ID No.4), 0.8 μM E-LF (SEQ ID No.5), 0.4 μM E-LB (SEQ ID No.6), 0.4 μM E-LMP (SEQ ID No.7), 0.2 μM UP2 (SEQ ID No.8);

[0112] 0.32 U / μL Bst 2.0 WarmStart ®The mixture consisted of DNA polymerase, 0.5 μL WarmStart RTx Reverse Transcriptase, 0.04 U / μL RNase HII, 40 mM guanidine hydrochloride, 5 μL genomic DNA, and the remainder was made up to 25 μL with nuclease-free water. The system was reacted at 60°C on a thermostat, with fluorescence collected every 1 minute for 60-90 cycles. In the above examples, the combination of endonuclease and mediator probe-universal probe (reporter probe) signal reporting mode enables specific analysis of dual target genes of the novel coronavirus within 90 minutes using a fluorescent probe system designed independently of the target sequence. The outer, inner, and circular primers used for detecting the N and E genes of the novel coronavirus in the above examples are not limited to the sequences provided in this invention. sequence list <110> Mengchao Hepatobiliary Hospital of Fujian Medical University (Fuzhou Infectious Disease Hospital) <120> A probe set, a probe set for detecting multiple nucleic acid targets of the novel coronavirus, a kit and detection method thereof <160> 17 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 tttcggaaga gacaggtac 19 <210> 2 <211> twenty two <212> DNA <213> Artificial Sequence <400> 2 aggaactcta gaagaattca ga 22 <210> 3 <211> 43 <212> DNA <213> Artificial Sequence <400> 3 cgcagtaagg atggctagtg tagcgtactt ctttttcttg ctt 43 <210> 4 <211> 43 <212> DNA <213> Artificial Sequence <400> 4 tcgattgtgt gcgtactgct gtttttaaca cgagagtaaa cgt 43 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <400> 5 actagcaaga ataccacga 19 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <400> 6 caatattgtt aacgtgagtc ttgtaaa 27 <210> 7 <211> 38 <212> DNA <213> Artificial Sequence <400> 7 tgggctctac gacctattgt taacgtgagt cttgtaaa 38 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <400> 8 ccgccggaac caggtcgtag agcccaccgg cgg 33 <210> 9 <211> 57 <212> DNA <213> Artificial Sequence <400> 9 tgggctctac gacctcgatt gtgtgcgtac tgctgttttt aacacgagag taaacgt 57 <210> 10 <211> 1260 <212> DNA <213> Artificial Sequence <400> 10 atgtctgata atggacccca aaatcagcga aatgcacccc gcattacgtt tggtggaccc 60 tcagattcaa ctggcagtaa ccagaatgga gaacgcagtg gggcgcgatc aaaacaacgt 120 cggccccaag gtttacccaa taatactgcg tcttggttca ccgctctcac tcaacatggc 180 aaggaagacc ttaaattccc tcgaggacaa ggcgttccaa ttaacaccaa tagcagtcca 240 gatgaccaaa ttggctacta ccgaagagct accagacgaa ttcgtggtgg tgacggtaaa 300 atgaaagatc tcagtccaag atggtatttc tactacctag gaactgggcc agaagctgga 360 cttccctatg gtgctaacaa agacggcatc atatgggttg caactgaggg agccttgaat 420 acaccaaaag atcacattgg cacccgcaat cctgctaaca atgctgcaat cgtgctacaa 480 cttcctcaag gaacaacatt gccaaaaggc ttctacgcag aagggagcag aggcggcagt 540 caagcctctt ctcgttcctc atcacgtagt cgcaacagtt caagaaattc aactccaggc 600 agcagtaggg gaacttctcc tgctagaatg gctggcaatg gcggtgatgc tgctcttgct 660 ttgctgctgc ttgacagatt gaaccagctt gagagcaaaa tgtctggtaa aggccaacaa 720 caacaaggcc aaactgtcac taagaaatct gctgctgagg cttctaagaa gcctcggcaa 780 840 caaacccaag gaattttgg ggaccaggaa ctaatcagac aaagactga ttacaaacat 900 tggccgcaaa ttgcacaatt tgcccccagc gcttcagcgt tcttcggaat gtcgcgcatt 960 ggcatggaag tcacaccttg gggaacgtgg ttgacctaca caggtgccat caaattggat 1020 gacaaagatc caaatttcaa agatcaagtc attttgctga ataagcatat tgacgcatac 1080 aaaacattcc caccaacaga gcctaaaaag gacaaaaaga agaaggtga tgaaactcaa 1140 gccttaccgc around gaagacaa actgtgactc ttcttcctgc tgcagatttg 1200 gatgatttct ccaaacaatt gcaacaatcc atgagcagtg ctgactcaac tcaggcctaa 1260 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <400> 11 gccaaaaggc ttctacgca 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 ttgctctcaa gctggttcaa 20 <210> 13 <211> 39 <212> DNA <213> Artificial Sequence <400> 13 tcccctactg ctgcctggag gcagtcaagc ctcttctcg 39 <210> 14 <211> 42 <212> DNA <213> Artificial Sequence <400> 14 tctcctgcta gaatggctgg catctgtcaa gcagcagcaa ag 42 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <400> 15 tgaatttctt gaactgttgc gactacg 27 <210> 16 <211> twenty one <212> DNA <213> Artificial Sequence <400> 16 atggcggtga tgctgctctt g 21 <210> 17 <211> 33 <212> DNA <213> Artificial Sequence <400> 17 tgggctcatc gtgctgcggt gatgctgctc ttg 33

Claims

1. A probe set for detecting multiple nucleic acid targets of the novel coronavirus, comprising a primer set for detecting the N gene of SARS-CoV-2 and / or the primer set for detecting the E gene of SARS-CoV-2; The primer set for detecting the E gene of SARS-CoV-2 includes: The first probe set and the second probe set, and the outer primer of the E gene E-WF / N-WR, the inner primer of the E gene E-FIP / N-BIP, and the circular primer of the E gene E-LF / N-LB. The primer set for detecting the N gene of SARS-CoV-2 includes: a third probe set, and outer primers N-WF / N-WR, inner primers N-FIP / N-BIP, and circular primers N-LF / N-LB. The first probe set is a loop primer targeting the E gene of SARS-CoV-2, which includes a loop mediator probe E-LMP with a nucleotide sequence as shown in SEQ ID No. 7, and one of two reporter probes UP1 / UP2 as shown in SEQ ID No. 8 and 18, respectively. The 5' end of the reporter probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group. The second probe set, targeting the inner primer of the E gene of SARS-CoV-2, includes an inner mediator probe E-IMP with a nucleotide sequence as shown in SEQ ID No. 9, and one of two reporter probes UP1 / UP2 as shown in SEQ ID No. 8 and 18, respectively. The 5' end of the reporter probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group. The third probe set consists of a loop primer targeting the N gene of SARS-CoV-2, comprising a loop mediator probe N-LMP with a nucleotide sequence as shown in SEQ ID No. 17, and one of two reporter probes UP1 / UP2 as shown in SEQ ID No. 8 and 18, respectively. The 5' end of the reporter probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group. It also includes a reporter probe and at least one mediator sub-probe, wherein, The mediator probe comprises a mediator sequence, an oligonucleotide sequence, and a target-specific sequence from 5' to 3'. The oligonucleotide sequence and the target-specific sequence include sequences complementary to a partial control sequence of the nucleic acid target sequence of the novel coronavirus. The partial control sequence of the nucleic acid target sequence is a sequence amplified by extending along the nucleic acid target sequence of the novel coronavirus after complementary pairing with a circular primer or an inner primer in a loop-mediated isothermal amplification reaction. The mediator sequence includes sequences that are not complementary to the nucleic acid target sequence or the control sequence. One base in the oligonucleotide sequence is modified to be an RNA base, and the rest are DNA bases. The RNA base is located at the 2nd to 3rd base from the 5' end of the target-specific sequence. The reporter probe comprises, from 3' to 5', a capture sequence complementary to the mediator sequence or a portion thereof, and a template sequence, wherein the RNA bases and the target-specific sequences at the RNA base ends are not complementary to the reporter probe. The reporter probe is labeled with a reporter group at its 5' end and a quencher group at its 3' end. The signal emitted by the reporter probe when hybridizing with its complementary sequence is different from the signal emitted when it does not hybridize with its complementary sequence. The reporter group and the quencher group are spaced 10-80 nt or longer apart.

2. The probe assembly according to claim 1, characterized in that, The nucleic acid target sequence of the novel coronavirus is selected from the conserved gene sequence of the novel coronavirus.

3. The probe assembly according to claim 2, characterized in that, The nucleic acid target sequence of the novel coronavirus is selected from the conserved gene sequences of the SARS-CoV-2 virus: N gene and E gene.

4. The probe assembly according to claim 1, characterized in that, N gene outer primer: Primer N-WF: nucleotide sequence as shown in SEQ ID No. 11; Primer N-WR: Nucleotide sequence as shown in SEQ ID No. 12; N gene inner primer: Primer N-FIP: Nucleotide sequence as shown in SEQ ID No. 13; Primer N-BIP: Nucleotide sequence as shown in SEQ ID No. 14; N gene circular primers: Primer N-LF: nucleotide sequence as shown in SEQ ID No. 15; Primer N-LB: nucleotide sequence as shown in SEQ ID No.

16.

5. The probe assembly according to claim 1, characterized in that, E gene outer primers: Primer E-WF: nucleotide sequence as shown in SEQ ID No. 1; Primer E-WR: nucleotide sequence as shown in SEQ ID No. 2; E gene inner primer: Primer E-FIP: Nucleotide sequence as shown in SEQ ID No. 3; Primer E-BIP: Nucleotide sequence as shown in SEQ ID No. 4; E gene circular primer: Primer E-LF: Nucleotide sequence as shown in SEQ ID No. 5; Primer E-LB: Nucleotide sequence as shown in SEQ ID No.

6.

6. A kit for detecting multiple nucleic acid targets of the novel coronavirus, characterized in that, The solution includes a primer set according to any one of claims 1 to 4, wherein the primer set solution is a solution in which each primer or probe in the primer set is diluted to 0.05 to 1.6 μmol / L.

7. The reagent kit according to claim 6, characterized in that, It also includes 6 mmol MgSO4, 1.4 mmol dNTP (A / G / C / T), 0.32 U / μL Bst DNA polymerase, 0.5 μL reverse transcriptase, 0.04 U / μL RNase HII, 5 μL template gene, and 1×Isothermal Amplification Buffer. The total volume of the detection system of the kit is 25 μL, and the remaining volume is made up to 25 μL with nuclease-free water.

8. The kit according to claim 6 or 7, characterized in that, The kit includes a solution containing the primer set for detecting the N gene of SARS-CoV-2 as described in claim 6. The primer set solution comprises 0.2 μmol N-WF, 0.2 μmol N-WR, 1.6 μmol N-FIP, 1.6 μmol N-BIP, 0.8 μmol N-LF, 0.4 μmol N-LB, 0.4 μmol N-LMP, 0.2 μmol UP1 or UP2, and further includes 6 mmol MgSO4, 1.4 mmol dNTPs (A / G / C / T), 0.32 U / μL Bst DNA polymerase, 0.5 μL reverse transcriptase, 0.04 U / μL RNase HII, 5 μL template gene, and 1×Isothermal Amplification Buffer. The total volume of the detection system is 25 μL, with the remainder made up to 25 μL with nuclease-free water.

9. The kit according to claim 6 or 7, characterized in that, The kit includes a solution containing the primer set for detecting the E gene of SARS-CoV-2 as described in claim 6. The primer set solution comprises 0.2 μmol E-WF, 0.2 μmol E-WR, 1.6 μmol E-FIP, 0.8 μmol E-BIP, 0.8 μmol E-LF, 0.8 μmol E-LB, 0.8 μmol E-LMP, 0.2 μmol UP1 or UP2, and further includes 6 mmol MgSO4, 1.4 mmol dNTPs (A / G / C / T), 0.32 U / μL Bst DNA polymerase, 0.5 μL reverse transcriptase, 0.04 U / μL RNase HII, 5 μL template gene, and 1×Isothermal Amplification Buffer. The total volume of the detection system is 25 μL, with the remainder made up to 25 μL with nuclease-free water.

10. The kit according to claim 6 or 7, characterized in that, The solution includes a primer set containing the primer set for detecting the N gene of SARS-CoV-2 as described in claim 6 and a primer set for detecting the E gene of SARS-CoV-2. The primer set solution for detecting the N gene of SARS-CoV-2 is as follows: 0.05 μmol N-WF, 0.05 μmol N-WR, 0.4 μmol N-FIP, 0.4 μmol N-BIP, 0.2 μmol N-LF, 0.1 μmol N-LB, 0.1 μmol N-LMP, 0.2 μmol UP1 or UP2; The primer set for detecting the E gene of SARS-CoV-2 is: 0.2 μmol E-WF, 0.2 μmol E-WR, 1.6 μmol E-FIP, 1.6 μmol E-BIP, 0.8 μmol E-LF, 0.4 μmol E-LB, 0.4 μmol E-LMP, and 0.2 μmol UP1 or UP2. The kit also includes 6 mmol MgSO4, 1.4 mmol dNTPs (A / G / C / T), 0.32 U / μL Bst DNA polymerase, 0.5 μL reverse transcriptase, 0.04 U / μL RNase HII, 40 mmol guanidine hydrochloride, 5 μL template gene, and 1×Isothermal Amplification Buffer. The total volume of the kit's detection system is 25 μL, with the remaining volume brought to 25 μL with nuclease-free water.

11. A method for detecting multiple nucleic acid targets of the novel coronavirus for non-disease diagnostic purposes, characterized in that, Includes the following steps: S1. Sampling; S2. Using the kit described in any one of claims 6 to 10 and the obtained sample, prepare a LAMP reaction system, place it in a reaction tube, mix and centrifuge; S3. Place the reaction tube on a fluorescence spectrometer with a constant temperature function and react at 60~65℃. Collect fluorescence every 1 minute for 60~90 cycles. S4. Read the Ct value. The criteria for judging the test results are as follows: if the Ct value of a sample is ≤60, the sample is judged to be positive. If 60 < Ct value ≤ 75, and the amplification curve is a logarithmic amplification curve, the sample is judged as a suspected positive sample; otherwise, the sample is judged as negative. For suspected positive samples, retesting is performed. If the Ct value of the retested sample is ≤60, the suspected positive sample is judged to be positive; otherwise, the sample is judged to be negative. If a sample has no Ct value or a Ct value > 75, the sample is considered negative.

12. The detection method according to claim 11, characterized in that, In step S3, if the kit in step S2 contains only primers for detecting the N or E gene, the amplification is performed at 65°C; if the kit in step S2 contains primers for detecting both the N and E genes, the amplification is performed at 60°C.

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