Dual-target reverse transcription fluorescent PCR primers, probes, and kits for detecting the novel coronavirus SARS-CoV-2

Abstract

The present invention provides dual-target site reverse transcription fluorescent PCR primers, probes, and a kit for detecting the novel coronavirus SARS-CoV-2. By performing nucleic acid sequence alignment on the entire genome of the novel coronavirus SARS-CoV-2, three specific nucleic acid sequences W1, W2, and W3 in the viral N gene are found, and the sequences are shown as SEQ ID NOs. 1-3, respectively. Multiple pairs of primers and probes are designed for the three target sites, respectively. It is found that the dual-target site reverse transcription fluorescent PCR using a primer-probe combination designed for the W1 and W2 targets (sequences shown as SEQ ID NOs. 4-6 and SEQ ID NOs. 7-9, respectively) can specifically and sensitively detect the novel coronavirus SARS-CoV-2, with a detection sensitivity of less than 10 copies. There is no non-specific amplification of 22 common clinical respiratory pathogens, which can effectively avoid false negative results caused by single target site mutations and has good specimen detection capabilities.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and in particular to target sites for detecting the novel coronavirus SARS-CoV-2, and specific primers and probes for the target sites. The present invention also relates to a method and a kit for detecting the novel coronavirus SARS-CoV-2 using the primers and probes. Background Art

[0002] The novel coronavirus (SARS-CoV-2) is a newly detected human pathogenic coronavirus, first detected in January 2020. Clinical manifestations include systemic symptoms such as fever and fatigue, accompanied by a dry cough. Dysspnea is common in hospitalized patients; the vast majority of patients present with generally stable vital signs upon admission. Close monitoring is still required to prevent further infection. Six genome sequences of the virus were released between January 11 and 12, 2020. Comparison revealed that the sequences are closely related to those of the SARS virus, indicating that it is a variant of the SARS virus. Among these six sequences, there are mutations in the coronavirus N gene.

[0003] Traditional methods for detecting viral infections include microscopic examination and antigen testing. Microscopic examination involves cell culture, which is time-consuming and requires certain microscopic examination techniques. Antigen testing is mostly based on ELISA, and although it can quickly produce test results, its sensitivity is still relatively low. Currently, the detection of viral infections mainly relies on nucleic acid detection technologies such as PCR and whole genome sequencing, especially real-time fluorescence PCR (qPCR), which has extremely high sensitivity and specificity and can quickly and accurately obtain test results. Currently, SARS-CoV-2 infection cannot be identified and diagnosed through specific clinical symptoms. Nucleic acid detection technology is its most important technical means. Through rapid RT-qPCR technology, it can quickly and accurately identify those with suspected clinical symptoms. Rapid detection of SARS-CoV-2 can be achieved.

[0004] SARS-CoV-2 is classified as a new variant of SARS virus, but there is currently no commercial SARS-CoV-2 nucleic acid detection kit certified by CFDA. Some biotechnology companies have developed scientific research nucleic acid detection kits for SARS-CoV-2 detection. However, all current SARS-CoV-2 nucleic acid detection kits are based on traditional RT-qPCR detection technology, and the primers and probes used in their detection are all designed for a single pathogen nucleic acid sequence target site. It is well known that the nucleic acid sequence of the virus has a high degree of variation, and the frequency of single base mutations is about 10 -6 , much higher than 10 of bacteria -9 Therefore, for nucleic acid testing of viruses, single target sites are prone to producing false negative results due to mutations, leading to missed detections. Summary of the Invention

[0005] The present invention aims to establish a highly sensitive and specific dual-target RT-qPCR method for SARS-CoV-2 detection, as well as specific primers, probes, and a kit for this method. This method addresses the current gaps in novel coronavirus nucleic acid detection methods. Given the high variability of viral nucleic acids, the dual-target design significantly reduces the risk of missed detection of variant viruses by a single target.

[0006] This invention, through whole-genome sequencing and alignment of six SARS-CoV-2 strains, identified three conserved and species-specific nucleic acid sequences (W1-W3) on the N gene of SARS-CoV-2, as shown in SEQ ID NO. 1-3, respectively. This invention utilizes multiple sets of specific primer-probe combinations for detecting these three targets. From these combinations, four sets of specific primer-probe combinations with excellent detection performance were further screened, achieving detection limits of 10-100 copies for the target sites. This invention further designs dual-target reverse transcription fluorescent PCR (RT-qPCR) primers and probes for the detection of this SARS-CoV-2 virus, and provides a method and kit for detection based on these primers.

[0007] Specifically, in the first aspect, the present invention first provides target sequences for detecting the novel coronavirus SARS-CoV-2, whose nucleotide sequences are shown in SEQ ID NO. 1, and / or SEQ ID NO. 2, and / or SEQ ID NO. 1. These three highly specific target sequences are named W1, W2, and W3, respectively.

[0008] The present invention provides the use of the above-mentioned target sequences W1, and / or W2, and / or W3 in detecting SARS-CoV-2 virus.

[0009] Preferably, the present invention provides a target sequence combination for detecting the new coronavirus SARS-CoV-2, namely W1+W2, whose nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0010] In a second aspect, the present invention provides the use of a detection reagent for detecting the above-mentioned target sequence or target sequence combination in detecting the new coronavirus SARS-CoV-2.

[0011] In a third aspect, the present invention provides a specific primer-probe combination for detecting the novel coronavirus SARS-CoV-2, which is any one or more of the following specific primer-probe combinations:

[0012] (1) The primer sequences are as shown in SEQ ID NO. 4-5, and the probe sequence is as shown in SEQ ID NO. 6; or

[0013] (2) The primer sequences are shown in SEQ ID NO.7-8, and the probe sequence is shown in SEQ ID NO.9; or

[0014] (3) The primer sequences are shown in SEQ ID NO. 10-11, and the probe sequence is shown in SEQ ID NO. 12; or

[0015] (4) The primer sequences are shown in SEQ ID NO. 13-14, and the probe sequence is shown in SEQ ID NO. 15.

[0016] The specific primer-probe combination mentioned above falls within the scope of protection of the present invention.

[0017] Preferably, the present invention provides a specific primer-probe combination for detecting the novel coronavirus SARS-CoV-2, which consists of two specific primer-probe combinations: (1) the primer sequence is shown in SEQ ID NO.4-5 and the probe sequence is shown in SEQ ID NO.6; and (2) the primer sequence is shown in SEQ ID NO.7-8 and the probe sequence is shown in SEQ ID NO.9.

[0018] The probe's 5' end is labeled with a fluorescent group, and its 3' end is labeled with a quencher group. The fluorescent group is selected from, but is not limited to, FAM, VIC, or CY5, and the quencher group is selected from, but is not limited to, BHQ1 or MGB. In an embodiment of the present invention, the probe type is an MGB probe, and the fluorescent group is FAM.

[0019] Fourthly, the present invention provides a dual-target reverse transcription fluorescence detection kit for detecting the novel coronavirus SARS-CoV-2, the kit containing two specific primer-probe combinations, (1) primer sequences as shown in SEQ ID NO.4-5 and probe sequences as shown in SEQ ID NO.6; and (2) primer sequences as shown in SEQ ID NO.7-8 and probe sequences as shown in SEQ ID NO.9.

[0020] The above kit works by using the genomic RNA of the sample to be tested as a template and using the combination of the (1st) and (2nd) specific primers and probes to perform dual-target reverse transcription quantitative PCR. The results are determined based on the amplification curve and fluorescence signal.

[0021] Preferably, the 25 μl reaction system of the reverse transcription fluorescence quantitative PCR is:

[0022] ;

[0023] And / or, the reaction procedure of the reverse transcription fluorescence quantitative PCR is: 50°C for 15 min; pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 58-61°C for 30 s, for 45 cycles.

[0024] In a fifth aspect, the present invention provides a dual-target site reverse transcription fluorescence detection method for detecting the novel coronavirus SARS-CoV-2, which uses the genomic RNA of the sample to be tested as a template and simultaneously utilizes the specific primer probe combination of groups (1) and (2) above to perform dual-target site reverse transcription fluorescence quantitative PCR, and determines the result based on the amplification curve and the fluorescence signal.

[0025] Those skilled in the art will understand that for the purpose of the epidemic prevention department to grasp and compile first-hand epidemiological data, or for the purpose of the inspection and quarantine department to inspect and quarantine imported and exported food, daily necessities, and animal and plant products, it is necessary to quickly, accurately, and sensitively detect the new coronavirus SARS-CoV-2. Therefore, the present invention provides a dual-target site reverse transcription fluorescence detection method for detecting the new coronavirus SARS-CoV-2 for non-disease diagnosis purposes, using the genomic RNA of the sample to be tested as a template, and using the specific primer probe combination of the above-mentioned groups (1) and (2) to perform dual-target site reverse transcription fluorescence quantitative PCR, and determine the results based on the amplification curve and the fluorescence signal.

[0026] Whether for disease diagnosis or non-disease diagnosis purposes, when the kit or method of the present invention is used to detect a sample, an NTC control (no template control), a NEG (negative control), and a POS control (positive control) must be set up each time a sample is tested. These three controls play a decisive role in the interpretation of the results: Effective amplification: NTC (-), NEG (-), POS (+);

[0027] Invalid amplification: NTC (-), NEG (-), POS (-) indicate reagent failure;

[0028] Invalid amplification: NTC (-), NEG (+), POS (+) indicate sample contamination;

[0029] Invalid amplification: NTC (+), NEG (+), POS (+) indicate system contamination.

[0030] Only sample test results under the condition of effective control amplification can be trusted; otherwise the test needs to be repeated.

[0031] When the three controls are effectively amplified during the test, the sample result judgment criteria are as follows:

[0032] Specimens with a CT value of 36 or less were considered positive;

[0033] Specimens with a CT value greater than 38 were negative results;

[0034] Specimens with CT values ​​between 36 and 38 need to be repeated. If the CT value is still lower than 38 after repeated testing, it is judged as positive amplification; if it exceeds 38, it is judged as negative amplification.

[0035] The present invention, through the comparison of the reported 6 SARS-CoV-2 whole genome nucleic acid sequences, discovered 3 conserved and species-specific nucleic acid sequences (W1-W3) on the N gene of SARS-CoV-2, designed and selected 4 sets of corresponding specific RT-qPCR detection probes and primers (systems 1-4 in the embodiment), and carried out system optimization and specificity verification. Subsequently, through pairwise combination optimization and screening, a dual-target site RT-qPCR kit based on the combination of systems 1 and 2 (corresponding to the aforementioned specific primer probe combinations (1) and (2)) was finally established. The kit has a detection sensitivity of less than 10 copies for SARS-CoV-2 and no non-specific amplification for 22 common clinical respiratory pathogens, showing good specificity. It can effectively avoid false negative results caused by single target mutations, which may result in missed detection of variant changes; the detection of dual target sites can enhance the fluorescence detection signal and improve the sensitivity of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1A-1D The following are the specificity test diagrams of 4 sets of RT-qPCR detection systems formed by using 1-4 sets of primer probe combinations. The results show that except for the positive template, the other templates are not amplified. Figure 1A-1D The values ​​marked on the horizontal axis from left to right are arranged in an arithmetic progression, namely 2, 4, 6, 8...40, 42, 44. Figure 2 , Figure 3 Figure A Figure 4 The same description applies to all x-axis layouts.

[0037] Figure 2 This is a comparison chart of the detection results of the dual-target RT-qPCR detection system for positive templates with the same concentration.

[0038] Figure 3 Figure 2 shows the standard amplification curve for the dual-target RT-qPCR detection system. A is a schematic diagram of the standard curve, and B is a logarithmic plot of the standard curve.

[0039] Figure 4 The figure shows the comparison results of the dual-target RT-qPCR detection system and the single-target system for site mutations. A is a simulated SARS-CoV-2 nucleic acid template, and B is a simulated SARS-CoV-2 mutant strain nucleic acid template. DETAILED DESCRIPTION

[0040] The following examples further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0041] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0042] Example 1: Gene Sequence Alignment and Target Sequence Determination of the Novel Coronavirus SARS-CoV-2

[0043] Based on the latest published 6 complete genomes of SARS-CoV-2, nucleic acid sequence comparison was performed through the NCBI database, and specific nucleic acid sequences in the N gene of the virus were found: W1 (SEQ ID NO.1), W2 (SEQ ID NO.2) and W3 (SEQ ID NO.3).

[0044] Example 2 Design of primers and probes for target sequences

[0045] For the target sequences identified in Example 1 (SEQ ID NOs. 1-3), the inventors designed multiple primer and probe combinations. From these numerous primer and probe combinations, four MGB probe and primer combinations with excellent detection performance were selected, as shown in Tables 1-4. System 1 was used to detect target sequence W1, system 2 was used to detect target sequence W2, and systems 3 and 4 were used to detect target sequence W3, respectively.

[0046] Table 1 Primer and probe sequences of system 1

[0047]

[0048] Table 2 Primer and probe sequences of system 2

[0049]

[0050] Table 3 Primer and probe sequences of system 3

[0051]

[0052] Table 4 Primer and probe sequences of system 4

[0053]

[0054] This example further optimized the annealing temperature of the four screened fluorescent quantitative PCR methods, varying it from 55°C to 65°C. The results showed that the optimal annealing temperature for system 1 was 58°C to 61°C, the optimal annealing temperature for system 2 was 56°C to 62°C, the optimal annealing temperature for system 3 was 58°C to 60°C, and the optimal annealing temperature for system 4 was 58°C to 62°C.

[0055] NTC control (no template control), NEG (negative control) and POS control (positive control) must be set up every time a sample is tested. These three controls play a decisive role in the interpretation of the results: Effective amplification: NTC (-), NEG (-), POS (+);

[0056] Invalid amplification: NTC (-), NEG (-), POS (-) indicate reagent failure;

[0057] Invalid amplification: NTC (-), NEG (+), POS (+) indicate sample contamination;

[0058] Invalid amplification: NTC (+), NEG (+), POS (+) indicate system contamination.

[0059] Only sample test results under the condition of effective control amplification can be trusted; otherwise the test needs to be repeated.

[0060] When the three controls are effectively amplified during the test, the sample result judgment criteria are as follows:

[0061] Specimens with a CT value of 36 or less were considered positive;

[0062] Specimens with a CT value greater than 38 were negative results;

[0063] Specimens with CT values ​​between 36 and 38 need to be repeated. If the CT value is still below 38 in the repeated test, it is considered a positive amplification; if it is above 38, it is considered a negative amplification.

[0064] Example 3: Validation of the sensitivity and specificity of four fluorescent PCR detection systems for SARS-CoV-2.

[0065] 1. Sensitivity evaluation

[0066] Due to the current lack of SARS-CoV-2 strains, sensitivity testing used synthetic SARS-CoV-2 sequences reverse-transcribed in vitro as positive templates. The synthetic SARS fragments, S1-S3, correspond to W1-W3 of SARS-CoV-2, respectively, and the sequences of S1-S3 are shown in SEQ ID NOs. 16-18, respectively; the synthetic MERS fragments, M1-M3, correspond to W1-W3 of SARS-CoV-2, respectively, and the sequences of M1-M3 are shown in SEQ ID NOs. 19-21, respectively. The synthetic sequences were spiked into 12 normal human throat swabs, and all four RT-qPCR tests were positive, with a sensitivity of 100% (12 / 12).

[0067] 2. Specificity evaluation

[0068] The four RT-qPCR assays screened and determined in Example 2 were used to detect 22 common respiratory strains and human chromosomes (see Table 5). The RNA sequences (corresponding to W1-W3) reverse transcribed from the artificially synthesized SARS-CoV-2 sequence in vitro were used as positive controls. The results showed that, except for the positive control, the remaining pathogen templates were negative (see Table 5). Figure 1A-1D ).

[0069] Table 5 Specific templates for reverse transcription fluorescence PCR system for detecting SARS-CoV-2

[0070]

[0071] 3. Evaluation of system detection limit

[0072] Using RNA sequences reverse-transcribed in vitro from synthetic SARS-CoV-2 sequences as templates, the detection limits of four RT-qPCR assays were evaluated. 5 μl of each concentration gradient was assayed, and real-time PCR was performed using the optimized reaction system and conditions, with triplicate replicates for each concentration gradient. The detection limits for system 1 were 10 copies, system 2 10–100 copies, system 3 100 copies, and system 4 10–100 copies.

[0073] Example 4 RT-qPCR system combination optimization and screening

[0074] The above four RT-qPCR detection systems were combined and optimized in pairs to form six dual-target detection combination systems: 1+2, 1+3, 1+4, 2+3, 2+4, and 3+4. These were compared with the most sensitive single-target system 1 (10 copies detection limit). The optimal dual-target system was the 1+2 system, with a detection limit of 10 copies. Compared with the most sensitive single-target system 1, its fluorescence signal value was higher and the detection ct value was lower, which was superior to the single-target system (see Figure 2 ). The dual-target RT-qPCR detection system of system 1+2 was determined to be the best solution for detecting SARS-CoV-2.

[0075] Example 5 Dual-target RT-qPCR detection system (system 1+2) and amplification conditions

[0076] The configuration of a 25 μl dual-target RT-qPCR detection system is as follows:

[0077] 25×Onestep RT-qPCR RTase mix 1.0ul

[0078] 5×Onestep RT-qPCR Buffer 5.0ul

[0079] PrimersARS-CoV-2-F1 (25uM) 0.2ul

[0080] PrimersARS-CoV-2 -R1 (25uM) 0.3ul

[0081] PrimersARS-CoV-2-P1 (25uM) 0.1ul

[0082] PrimersARS-CoV-2-F2(25uM) 0.2ul

[0083] PrimersARS-CoV-2-R2(25uM) 0.2ul

[0084] PrimersARS-CoV-2-P2(25uM) 0.1ul

[0085] Nuclease-Free Water (Promega P1193) 12.9ul

[0086] Template 5.0ul

[0087] Amplification conditions: 50°C for 15 min; pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 58-61°C for 30 s, 45 cycles.

[0088] Example 6 Standard Curve of Dual-Target RT-qPCR Detection System

[0089] The real-time PCR standard curve of the dual-target RT-qPCR detection system determined in Example 4 was drawn with the log value of the standard concentration template as the horizontal axis and the corresponding Ct value as the vertical axis. The results showed that the positive template amount was between 10-10 6 When the number of copies is within this range, the logarithm value has a very good correlation with the Ct value (R 2 =1.000), and the amplification efficiency E=93.4% (see Figure 3 ).

[0090] Example 7: Specificity Validation of the Dual-Target RT-qPCR Detection System

[0091] The dual-target RT-qPCR detection method identified in Example 4 was used to detect 22 common respiratory strains and human chromosomes (see Table 5), using RNA sequences reverse-transcribed in vitro from the synthetic SARS-CoV-2 sequence as a positive control. The results were negative for all pathogen templates except the positive control, demonstrating that the dual-target RT-qPCR detection method identified in this invention has a specificity of 100%.

[0092] Example 8 Simulating the Effect of Single-Base Mutations in SARS-CoV-2 on the Detection Results of the Dual-Target RT-qPCR Detection System and the Single-Target System (System 1)

[0093] Based on the specific sequence fragment W1 in the N gene of SARS-CoV-2 determined in Example 1, the virus mutation in vitro was simulated and a variant sequence fragment W1' with a single base mutation at the probe site was designed (the T base at position 34 relative to the W1 sequence was mutated to a G base). The W1+W2 sequence was mixed to simulate the existing SARS-CoV-2 nucleic acid template, and the W1'+W2 sequence was mixed to simulate the SARS-CoV-2 single base mutant nucleic acid template. The dual-target RT-qPCR detection system (system 1+2) and the single-target system (system 1) were tested respectively. 10 3 The dual-target RT-qPCR detection system and the single-target system can detect the simulated SARS-CoV-2 nucleic acid template (W1+W2 sequence mixture) ( Figure 4 A), only the dual-target RT-qPCR detection system can detect the simulated SARS-CoV-2 nucleic acid template (W1'+W2 sequence mixture) (see Figure 4 B).

[0094] In summary, the dual-target RT-qPCR detection system (System 1+2) provided by the present invention achieves good specificity, excellent sensitivity, and high accuracy in detecting SARS-CoV-2: detection sensitivity is within 10 copies, and there is no nonspecific amplification for 22 common clinical respiratory pathogens, demonstrating good specificity. The identification of dual targets, primer and probe design, and simultaneous testing of test samples can effectively avoid false negative results caused by single-target mutations in the virus, which can lead to missed detections. This system meets the requirements of clinical testing and food hygiene inspection and quarantine for rapid detection of the novel coronavirus SARS-CoV-2, achieving excellent results in these fields and promising application prospects.

[0095] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein. Sequence Listing <110> Xi'an Broadcorn Biomedical Center Co., Ltd. <120> Dual-target reverse transcription fluorescent PCR primers, probes, and kits for detecting the novel coronavirus SARS-CoV-2 <130> KHP201110350.3 <160> twenty one <170> SIPOSequenceListing 1.0 <210> 1 <211> 71 <212> DNA <213> Artificial Sequence <400> 1 tggctggcaa tggcggtgat gctgctcttg ctttgctgct gcttgacaga ttgaaccagc 60 ttgagagcaa a 71 <210> 2 <211> 83 <212> DNA <213> Artificial Sequence <400> 2 tctaagaagc ctcggcaaaa acgtactgcc actaaagcat acaatgtaac acaagctttc 60 ggcagacgtg gtccagaaca aac 83 <210> 3 <211> 107 <212> DNA <213> Artificial Sequence <400> 3 cgcattggca tggaagtcac accttcggga acgtggttga cctacacagc tgccatcaaa 60 ttggatgaca aagatccaaa tttcaaagat caagtcattt tgctgaa 107 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <400> 4 tggcaatggc ggtgatg 17 <210> 5 <211> twenty two <212> DNA <213> Artificial Sequence <400> 5 agctggttca atctgtcaag ca 22 <210> 6 <211> 16 <212> DNA <213> Artificial Sequence <400> 6 tgctcttgct ttgctg 16 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <400> 7 gaagcctcgg caaaaacg 18 <210> 8 <211> twenty two <212> DNA <213> Artificial Sequence <400> 8 gccgaaagct tgtgttacat tg 22 <210> 9 <211> 16 <212> DNA <213> Artificial Sequence <400> 9 actgccacta aagcat 16 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <400> 10 cgggaacgtg gttgaccta 19 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <400> 11 tctttgaaat ttggatcttt gtcatc 26 <210> 12 <211> 14 <212> DNA <213> Artificial Sequence <400> 12 cagstgccat caaa 14 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 agtcacacct tcgggaacgt 20 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <400> 14 tctttgaaat ttggatcttt gtcatc 26 <210> 15 <211> 17 <212> DNA <213> Artificial Sequence <400> 15 ttgacctaca cagstgc 17 <210> 16 <211> 71 <212> DNA <213> Artificial Sequence <400> 16 tggctagcgg aggtggtgaa actgccctcg cgctattgct gctagacaga ttgaaccagc 60 ttgagagcaa a 71 <210> 17 <211> 83 <212> DNA <213> Artificial Sequence <400> 17 tctaaaaagc ctcgccaaaa acgtactgcc acaaaacagt acaacgtcac tcaagcattt 60 gggagacgtg gtccagaaca aac 83 <210> 18 <211> 107 <212> DNA <213> Artificial Sequence <400> 18 cgcattggca tggaagtcac accttcggga acatggctga cttatcatgg agccattaaa ttggatgaca aagatccaca attcaaagac aacgtcatac tgctgaa <210> 19 <211> 71 <212> DNA <213> Artificial Sequence <400> 19 tcggagcagt aggaggtgat ctactttacc ttgatcttct gaacagacta caagcccttg agtctggcaa to <210> 20 <211> 77 <212> DNA <213> Artificial Sequence <400> 20 60. aatagatgc gccacaagcg cacttccacc aaaagtttca acatggtgca agcttttggt cttcgcggac wealth <210> 21 <211> 108 <212> DNA <213> Artificial Sequence <400> 21 60. cgggctgtta aggatggcat cgtttgggtc catgaagatg gcgccactga tgctccttca acttttggga cgcggaaccc ttacaatgat tcagctattg ttacacaa

Claims

1. A specific primer-probe combination for detecting the novel coronavirus SARS-CoV-2, which is any one or more of the following specific primer-probe combinations: (1) The primer sequences are shown in SEQ ID NO. 4-5, and the probe sequence is shown in SEQ ID NO. 6; or (2) The primer sequences are shown in SEQ ID NO.7-8, and the probe sequence is shown in SEQ ID NO.9; or (3) The primer sequences are shown in SEQ ID NO. 10-11, and the probe sequence is shown in SEQ ID NO. 12; or (4) The primer sequences are shown in SEQ ID NO. 13-14, and the probe sequence is shown in SEQ ID NO.

15.

2. A specific primer-probe combination for detecting the novel coronavirus SARS-CoV-2, comprising two specific primer-probe combinations, (1) the primer sequences are shown in SEQ ID NOs. 4-5, and the probe sequences are shown in SEQ ID NO. 6; and (2) the primer sequences are shown in SEQ ID NOs. 7-8, and the probe sequences are shown in SEQ ID NO.

9.

3. A kit comprising the specific primer-probe combination according to claim 1.

4. A dual-target site reverse transcription fluorescence detection kit for detecting the new coronavirus SARS-CoV-2, characterized in that: Contains the specific primer-probe combination according to claim 2.

5. The kit according to claim 4, wherein Using the genomic RNA of the sample to be tested as a template, the (1st) and (2nd) specific primer probe combinations are used to perform dual-target site reverse transcription fluorescence quantitative PCR, and the results are determined based on the amplification curve and fluorescence signal.

6. The kit according to claim 5, wherein The 25 μl reaction system for reverse transcription fluorescence quantitative PCR is: And / or, the reaction procedure of the reverse transcription fluorescence quantitative PCR is: 50°C for 15 min; pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 58-61°C for 30 s, for 45 cycles.

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