WO2023159572A1

WO2023159572A1 - Detection kit, primer composition and detection method for novel coronavirus sars-cov-2

Info

Publication number: WO2023159572A1
Application number: PCT/CN2022/078307
Authority: WO
Inventors: 颜菁; 袁青; 刘艳
Original assignee: 江苏汇先医药技术有限公司
Priority date: 2022-02-28
Filing date: 2022-02-28
Publication date: 2023-08-31

Abstract

Provided in the present invention are a detection kit, primer composition and detection method for novel coronavirus SARS-CoV-2. The detection kit for novel coronavirus SARS-CoV-2 comprises a first primer set for an Orflab gene of novel coronavirus SARS-CoV-2 and a second primer set for an N gene of novel coronavirus SARS-CoV-2; the first primer set comprises outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, loop primer LF and loop primer LB having nucleotide sequences sequentially as shown in SEQ ID NO. 1 to SEQ ID NO. 6; and the second primer set comprises outer primer F3, outer primer B3, inner primer FIP, inner primer BIP and loop primer LB having nucleotide sequences sequentially as shown in SEQ ID NO. 7 to SEQ ID NO. 11. The detection kit, primer composition and detection method for novel coronavirus SARS-CoV-2 of the present invention have high detection accuracy, good sensitivity, convenient operation and short detection time.

Description

Detection kit, primer composition and detection method of novel coronavirus SARS-CoV-2

technical field

The invention belongs to the field of detection kits, and relates to a detection kit, a primer composition, and a detection method for novel coronavirus SARS-CoV-2, in particular to a detection reagent for detecting novel coronavirus SARS-CoV-2 based on a LAMP method box.

Background technique

Novel coronavirus pneumonia (COVID-19) is an acute respiratory infectious disease caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2 or 2019-nCoV) infection, mainly lung lesions, clinical manifestations are fever , fatigue, dry cough as the main manifestations, nasal congestion, runny nose and other upper respiratory symptoms are rare. About half of the patients developed dyspnea more than a week later, and severe cases rapidly progressed to acute respiratory distress syndrome, septic shock, metabolic acidosis that was difficult to correct, and coagulation dysfunction.

At present, the in vitro nucleic acid detection of the new coronavirus is mainly used to judge whether it is infected with the new coronavirus. Fluorescence RT-PCR method, which is widely used at present, although this method is widely used now, has certain deficiencies in detection accuracy and sensitivity, and requires large precision instruments and professional technicians to operate, which invisibly causes detection Due to the long time and high cost of detection, the detection of the new coronavirus can only be completed in professional genetic testing institutions or tertiary hospitals, and it cannot yet become an on-site instant detection method for the new coronavirus, which cannot effectively alleviate the current shortage of new coronavirus detection. question.

Contents of the invention

For above-mentioned technical problem, the object of the present invention is to provide a kind of detection kit and primer composition, detection method of novel coronavirus SARS-CoV-2, while its detection accuracy rate is higher, sensitivity is better and easy to operate, detection The time is shorter.

According to the first aspect of the present invention, a detection kit for novel coronavirus SARS-CoV-2 includes the first primer set for the Orflab gene of the novel coronavirus SARS-CoV-2 and the primer set for the novel coronavirus SARS-CoV-2. The second primer set of the N gene of the virus SARS-CoV-2; the first primer set includes the outer primer F3, outer primer B3, outer primer B3, Inner primer FIP, inner primer BIP, loop primer LF and loop primer LB; the second primer set includes outer primer F3 and outer primer B3 whose nucleotide sequences are sequentially shown in SEQ ID NO.7～SEQ ID NO.11 , inner primer FIP, inner primer BIP, and loop primer LB.

The nucleotide sequence of the primer for the Orflab gene of SARS-CoV-2:

F3: 5'-TGTTTTAAGCGGACACAATC-3', SEQ ID NO.1;

B3: 5'-GAACAGTATCTACACAAACTCTT-3', SEQ ID NO.2;

FIP: 5'-AGCACTCTTAAGAAGTTGAATGTCTTGCTAAACACTGTCTTCATG-3', SEQ ID NO.3;

BIP: 5'-AATCAGCACGAAGTTCTACTTGCTGTATAGGGTCAGCACCAA-3', SEQ ID NO.4;

LF: 5'-TCACCTTTGTTAACATTTGGGCC-3', SEQ ID NO.5;

LB: 5'-ACCATTATTATCAGCTGGTATTT-3', SEQ ID NO.6.

The nucleotide sequence of the primer for the N gene of SARS-CoV-2:

F3: 5'-CGGCAGTCAAGCCTCTTC-3', SEQ ID NO.7;

B3: 5'-TTGCTCTCAAGCTGGTTCAA-3', SEQ ID NO.8;

FIP: 5'-TCCCCTACTGCTGCCTGGAGCGTTCCTCATCACGTAGTCG-3', SEQ ID NO.9;

BIP: 5'-TTCTCCTGCTAGAATGGCTGGCTCTGTCAAAGCAGCAGCAAAG-3', SEQ ID NO.10;

LB: 5'-GCGGTGATGCTGCTCTTG-3', SEQ ID NO.11.

In a preferred embodiment, each primer is individually packaged. Dilute and mix before use. More preferably, the molar ratio of the primers Orflab-F3, Orflab-B3, Orflab-FIP, Orflab-BIP, Orflab-LF and Orflab-LB of the first primer set is preferably 1:1:8:8:4:4, It has a good amplification effect on the Orflab gene, obtains the lowest detection limit on the Orflab gene, and improves the accuracy of the detection result. The molar ratio of the primers N-F3, N-B3, N-FIP, N-BIP and N-LF of the second primer set is preferably 1:1:8:8:4, which has better amplification of the N gene The effect is to obtain the lowest detection limit for the N gene and improve the accuracy of the detection results. When in use, prepare the primer mixture, specifically: dissolve FIP, BIP, B3, F3, LF, and LB into solutions respectively, according to F3:B3:FIP:BIP:LF:LB=1:1:8:8: Mix at a ratio of 4:4, if there is no LF primer, make up with ddH20.

In a preferred embodiment, each primer of the first primer set forms the first primer mixture; each primer of the second primer set forms the second primer mixture.

More preferably, in the first primer mixture, the molar ratio of the outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, loop primer LF, and loop primer LB of the first primer set is 1:1 :8:8:4:4; in the second primer mixture, the molar ratio of the outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, and loop primer LB of the second primer set is 1 :1:8:8:4.

Further, the detection kit also includes a combination of one or more of the following substances: dNTPs, MgCl ₂ , 10×Isothermo Buffer, enzyme, 20×EvaGreen Dye, template and ddH ₂ 0.

Further, the kit also includes a positive quality control product, and the positive quality control product includes an armored virus containing a target fragment of the Orflab gene and an armored virus containing a target fragment of the N gene.

Further, the template includes an armored virus containing a target fragment of an Orflab gene and an armored virus containing a target fragment of an N gene.

In a preferred embodiment, the interpretation standard of the detection kit is: if the fluorescence curves for the Orflab gene and the N gene all have "S" shaped curves, it is judged as positive; if the fluorescence curves for the Orflab gene and the N gene If neither of the curves has an "S"-shaped curve, it is judged as negative; if one of the fluorescence curves for the Orflab gene and the N gene has an "S"-shaped curve and the other does not have an "S"-shaped curve, re-test, if still If one has an "S"-shaped curve and the other does not have an "S"-shaped curve, it is judged as positive.

According to a second aspect of the present invention, a primer composition for a novel coronavirus SARS-CoV-2, comprising a first primer set directed at the Orflab gene of the novel coronavirus SARS-CoV-2 and directed against the novel coronavirus The second primer set of the N gene of the virus SARS-CoV-2; the first primer set includes the outer primer F3, outer primer B3, outer primer B3, Inner primer FIP, inner primer BIP, loop primer LF and loop primer LB; the second primer set includes outer primer F3 and outer primer B3 whose nucleotide sequences are sequentially shown in SEQ ID NO.7～SEQ ID NO.11 , inner primer FIP, inner primer BIP, and loop primer LB.

According to the third aspect of the present invention, a method for detecting the novel coronavirus SARS-CoV-2 for non-diagnostic and therapeutic purposes, using the above-mentioned detection kit or the above-mentioned primer composition, after mixing with the sample, A loop-mediated isothermal amplification reaction was performed.

In a preferred embodiment, the procedure of the amplification reaction is to react at 60-65°C for 18-30 minutes. More preferably, the procedure of the amplification reaction is to react at 65°C for 20 minutes.

In a preferred embodiment, the first amplification system is used to detect the Orflab gene in the sample, wherein: the first amplification system includes the first primer set, dNTPs, MgCl ₂ , 10×Isothermo Buffer, enzyme, 20×EvaGreen Dye, template and ddH ₂ 0. More preferably, the template comprises an armored virus containing a target segment of the Orflab gene.

In a preferred embodiment, the second amplification system is used to detect the N gene in the sample, wherein: the second amplification system includes the second primer set, dNTPs, MgCl ₂ , 10×Isothermo Buffer, enzyme, 20 ×EvaGreen Dye, template and ddH ₂ 0. More preferably, the template comprises an armored virus containing a target segment of the N gene.

More preferably, in the first amplification system or the second amplification system, the concentration of the primer composition is 0.2-2 μM, the concentration of dNTPs is 1.0-2.0 mM, and the concentration of MgCl ₂ is 5-10 mM.

More preferably, the molar ratio of the outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, loop primer LF and loop primer LB of the first primer set is 1:1:8:8:4:4, The molar ratio of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP, and the loop primer LB of the second primer set is 1:1:8:8:4.

Specifically, during detection, samples are respectively added to the first amplification system and the second amplification system, and loop-mediated isothermal amplification is performed respectively, so as to simultaneously detect whether the samples contain the Orflab gene and the N gene.

The present invention adopts the above scheme, and has the following advantages compared with the prior art:

The detection kit, primer composition, and detection method of the novel coronavirus SARS-CoV-2 of the present invention are detected based on the loop-mediated isothermal amplification method, and are respectively aimed at 6 of the Orflab gene and the N gene of the SARS-CoV-2 virus. Design 4 specific primers for each region, add 2 or 1 loop primers, and perform amplification in a constant temperature environment of 60-65°C, with good sensitivity (the lowest detection limit can reach 10copies/mL and 50copies/mL) , the operation is simple, and the time is short; and it can detect the multi-gene loci of the SARS-CoV-2 virus at the same time, which improves the accuracy of the detection; and does not rely on expensive professional equipment, and can be operated by ordinary personnel without specialization , providing the possibility for on-site instant detection of SARS-CoV-2 virus.

Description of drawings

In order to illustrate the technical solution of the present invention more clearly, the accompanying drawings that need to be used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. Ordinary technicians can also obtain other drawings based on these drawings on the premise of not paying creative work.

Fig. 1 is the minimum detection limit experimental result figure of the SARS-CoV-2 virus Orflab gene target of the kit of embodiment 2;

Fig. 2 is the minimum detection limit experimental result graph of the SARS-CoV-2 virus N gene target of the kit of Example 2.

Detailed ways

The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art. It should be noted here that the descriptions of these embodiments are used to help understand the present invention, but are not intended to limit the present invention.

Reagents and materials required for the examples: primers were synthesized by General Biosystems (Anhui) Co., Ltd.; dNTPs were purchased from Suo Laibao Biotechnology Co., Ltd.; nucleic acid dye 20×EvaGreen Dye was purchased from biotium; new crown orf1ab and N gene pseudovirus Synthesized by Shanghai Kamin Biotechnology Co., Ltd.

Example 1: Design and Screening of Respiratory Syncytial Virus Primer Compositions

This embodiment designs and screens the primers for detecting the new coronavirus SARS-CoV-2 based on RT-LAMP, specifically including the RT-LAMP primer set for detecting the Orflab gene of the new coronavirus SARS-CoV-2 and detecting the new coronavirus SARS-CoV-2 The RT-LAMP primer set for the N gene, the specific process is described below.

Through the SARS-CoV-2 genome downloaded in GISAID, NCBI downloaded other common coronaviruses and used them to design RT-LAMP primer sets. Specifically: Viral HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV sequences, BioEdit and SnapGene sequence comparison analysis, to obtain specific Orflab fragments and N fragment sequences.

(1) Using the online design software Primer ExplorerV5, design RT-LAMP primers for the specific Orflab gene fragment and N gene fragment, design 4 sets of primers for the Orflab gene fragment, and design 5 sets of primers for the N gene fragment.

(2) The armored virus containing the Orflab target fragment was serially diluted with ddH ₂ 0, and the copy numbers were 10 ⁶ copies/mL, 10 ⁵ copies/mL, 10 ⁴ copies/mL, and 10 ³ copies/mL. The armored virus containing the N gene target fragment was serially diluted with ddH ₂ 0, and the copy numbers were 10 ⁶ copies/mL, 10 ⁵ copies/mL, 10 ⁴ copies/mL, and 10 ³ copies/mL.

(3) Amplification reaction system: amplification primer Mix 2.2μL, dNTPs 1.0-2.0mM, MgCl2: 5-10mM, 10×Isothermo Buffer 2.5μL, enzyme Mix 0.2μL, 20X EvaGreen Dye 1μL, template 5μL, add ddH20 to make up to 25 μL, the amplification primer reaction program is 65° C. for 20 min. The template includes two types, the template for the amplification reaction system of the Orflab gene is an armored virus containing the target fragment of the Orflab gene, and the template for the amplification reaction system of the Orflab gene is an armored virus containing the target fragment of the N gene.

See Table 1 for Orflab gene primer information.

Table 1

See Table 2 for N gene primer information.

After the amplification reaction is over, judge whether it is positive according to whether the fluorescence curve is an "S" curve. An "S" curve indicates that the test result is positive, and no amplification curve indicates that the test result is negative. The amplification results are shown in Table 3.

Table 3 Orf1ab gene and N gene primer screening amplification results

It can be seen from Table 3 that the primers with the best amplification effect of the Orf1ab gene are the primers of the second group, and the primers of the third group are the best with the amplification effect of the N gene.

Embodiment 2: detection kit

The detection kit includes: first primer set, second primer set, dNTPs, MgCl ₂ , 10×Isothermo Buffer, enzyme, 20×EvaGreen Dye, ddH ₂ 0. The first primer set is the second primer set in Table 1, the second primer set is the third primer set in Table 2, and each primer is packaged independently. The kit also includes templates used as positive quality controls, including two armored viruses containing Orflab gene and N gene target fragments as templates, which were synthesized by Shanghai Kamin Biotechnology Co., Ltd.

Before use, prepare the amplification reaction system, including: amplification primer Mix 2.2 μL, dNTPs 1.4 mM, MgCl ₂ 8 mM, 10×Isothermo Buffer 2.5 μL, enzyme Mix 0.2 μL, 20X EvaGreen Dye 1 μL, template 5 μL, add ddH20 Make up to 25 μL. Among them, two amplification reaction systems need to be prepared; the amplification primer Mix in the first amplification reaction system is a mixture of primers F3, B3, FIP, BIP, LF and LB for the Orflab gene, and the template is a mixture containing the Orflab gene target The fragmented armored virus; the amplification primer Mix in the second amplification reaction system is a mixture of primers F3, B3, FIP, BIP, LF and LB for the N gene, and the template is the armored virus containing the N gene target fragment.

In the first primer set detecting the Orflab gene of the new coronavirus SARS-CoV-2, the molar ratio of the primers Orflab-F3, Orflab-B3, Orflab-FIP, Orf1ab-BIP, Orflab-LF and Orflab-LB is preferably 1: 1:8:8:4:4. In the second primer set for detecting the N gene of the new coronavirus SARS-CoV-2, the molar ratio of the primers N-F3, N-B3, N-FIP, N-BIP and N-LF is preferably 1:1:8: 8:4. The primer mixture was prepared according to the molar ratio of the above primers. Prepare the amplification primer Mix, dissolve FIP, BIP, B3, F3, LF, and LB into a 100umol/L solution, according to F3:B3:FIP:BIP:LF:LB=1:1:8:8:4:4 If there is no LF primer, use ddH20 to make up.

The method of using the kit is as follows:

(1) Sample processing: Take a sample (such as a throat swab or oral swab sample) and transfer it to a centrifuge tube containing the sample lysate, shake and mix well, and wait for inspection;

(2) Add the first amplification system (20 μL) and the liquid (5 μL) in step (1) to test tube 1, add the second amplification system (20 μL) and the liquid in step (1) to test tube 2 Liquid (5 μL), react in a nucleic acid detector at 65°C for 20 minutes;

(3) Interpretation of results: According to whether the fluorescence curve is an "S" curve to determine whether it contains the new coronavirus, an "S" curve indicates that the test sample contains the new coronavirus. In this example, if the two targets of the Orflab gene of the new coronavirus are consistent, it will be judged according to the consistent result; if one of the two targets is positive and the other is negative, the test will be repeated, and if the test result is still one negative and one positive, then it will be judged as positive. determination.

Feasibility of the kit

1. Lower limit of detection (LOD) experiment

The armored virus containing the Orflab target fragment and the armored virus containing the N gene target fragment were serially diluted with ddH ₂ 0, and the copy numbers were 10 ⁵ copies/mL, 10 ⁴ copies/mL, 10 ³ copies/mL, 100 copies/mL, 50copies/mL, 10copies/mL. Use the kit of Example 2 to detect the Orflab target template according to the method described in Example 2, use the kit of Example 2 to detect the N gene target template according to the method described in Example 2, and determine the minimum detection method for positive nucleic acids quantity. As shown in Figure 1, the minimum detection limit of the nucleic acid of the Orflab gene target of the new coronavirus SARS-CoV-2 is 10copies/mL; as shown in Figure 2, the minimum detection limit of the nucleic acid of the N gene target of the new coronavirus SARS-CoV-2 is 50copies /mL.

2. Specificity experiment

(1) Extraction of specific experimental pathogen samples: Influenza A virus (H1N1), influenza B virus (Yamagata), respiratory syncytial virus (type A), adenovirus (type 2), parainfluenza virus (type 1) , Middle East Respiratory Syndrome Coronavirus (MERSr-CoV), Human Coronavirus HCoV-229E, Human Coronavirus HCoV-HK U1, Human Coronavirus HCoV-NL63, Human Coronavirus HCoV-0C43, Influenza A Virus (H3N2) 13 viruses, Influenza A virus (H5N1) and Influenza A virus (H7N9), were purified by the RNA virus genome extraction kit of Suleibao Biotechnology Co., Ltd. respectively.

(2) Sample detection: The kits in Example 2 were used to detect the above 13 viral nucleic acids, the Orflab gene target and N gene target templates were used as positive controls, and ddH ₂ 0 was used as a negative control.

(3) Analysis of results: see Table 4, Influenza A virus (H1N1), Influenza B virus (Yamagata), Respiratory syncytial virus (Type A), Adenovirus (Type 2), Parainfluenza virus (Type 1), Middle East respiratory syndrome coronavirus (MERSr-CoV), human coronavirus HCoV-229E, human coronavirus HCoV-HK U1, human coronavirus HCoV-NL63, human coronavirus HCoV-0C43, influenza A virus (H3N2), Influenza A virus (H5N1) and influenza A virus (H7N9) were not detected, indicating that the kit has high specificity.

Table 4 specificity experimental results

"+" means positive, and "-" means negative.

As shown in this specification and claims, the terms "comprising" and "comprising" only suggest the inclusion of explicitly identified steps and elements, and these steps and elements do not constitute an exclusive list, and the method or device may also include other steps or elements.

The singular forms "a", "said" and "the" are also intended to include the plural unless the context clearly dictates otherwise.

The above-described embodiment is only to illustrate the technical concept and characteristics of the present invention. It is a preferred embodiment. Its purpose is that those familiar with this technology can understand the content of the present invention and implement it accordingly, and cannot limit the scope of the present invention. protected range. All equivalent changes or modifications made according to the principles of the present invention shall fall within the protection scope of the present invention.

Claims

A detection kit for novel coronavirus SARS-CoV-2, characterized in that it includes the first primer set for the Orflab gene of the novel coronavirus SARS-CoV-2 and the primer set for the novel coronavirus SARS-CoV- The second primer set of the N gene of 2; the first primer set includes the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer F3 shown in SEQ ID NO. Primer BIP, loop primer LF and loop primer LB; the second primer set includes the outer primer F3, outer primer B3, inner primer FIP, Inner primer BIP, and loop primer LB.
The detection kit according to claim 1, wherein each primer is packaged separately.
The detection kit according to claim 1, wherein each primer of the first primer set forms a first primer mixture; each primer of the second primer set forms a second primer mixture.
The detection kit according to claim 3, wherein in the first primer mixture, the outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, and loop primer LF of the first primer set And the molar ratio of the loop primer LB is 1:1:8:8:4:4; in the second primer mixture, the outer primer F3, outer primer B3, inner primer FIP, inner primer The molar ratio of BIP and loop primer LB is 1:1:8:8:4.
The detection kit according to claim 4, wherein the detection kit further comprises a combination of one or more of the following substances: dNTPs, MgCl 2 , 10×Isothermo Buffer, enzyme, 20×EvaGreen Dye , template and ddH 2 0.
The detection kit according to claim 5, wherein the template comprises an armored virus containing an Orflab gene target fragment and an armored virus containing an N gene target fragment.
The detection kit according to claim 1, wherein the kit also includes a positive quality control product, and the positive quality control product includes an armored virus containing the Orflab gene target fragment and an armored virus containing the N gene target fragment .
The detection kit according to claim 1, wherein the interpretation standard of the detection kit is: if the fluorescence curves for the Orflab gene and the N gene all have an "S"-shaped curve, then it is judged as positive; Neither the fluorescence curves of the Orflab gene nor the N gene has an "S"-shaped curve, and it is judged as negative; if one of the fluorescence curves for the Orflab gene and the N gene has an "S"-shaped curve and the other has no "S"-shaped curve , then re-test, if one still has an "S"-shaped curve and the other does not have an "S"-shaped curve, it is determined to be positive.
A primer composition for novel coronavirus SARS-CoV-2, characterized in that it includes the first primer set for the Orflab gene of the novel coronavirus SARS-CoV-2 and the primer set for the novel coronavirus SARS-CoV- The second primer set of the N gene of 2; the first primer set includes the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer F3 shown in SEQ ID NO. Primer BIP, loop primer LF and loop primer LB; the second primer set includes the outer primer F3, outer primer B3, inner primer FIP, Inner primer BIP, and loop primer LB.
A method for detecting the novel coronavirus SARS-CoV-2 of non-diagnostic and therapeutic purposes, characterized in that the detection kit as claimed in any one of claims 1 to 8 or the primer composition as claimed in claim 7 are used , and after mixing with the sample, the loop-mediated isothermal amplification reaction is performed.
The detection method according to claim 10, characterized in that the procedure of the amplification reaction is to react at 60-65° C. for 10-30 minutes.
The detection method according to claim 10, characterized in that, use the first amplification system to detect the Orflab gene in the sample, and use the second amplification system to detect the N gene in the sample, wherein: the first amplification system includes The first primer set, dNTPs, MgCl 2 , 10×Isothermo Buffer, enzyme, 20×EvaGreen Dye, template and ddH 2 0; the second amplification system includes the second primer set, dNTPs, MgCl 2 , 10×Isothermo Buffer, enzyme, 20×EvaGreen Dye, template and ddH 2 0.
The detection method according to claim 12, characterized in that, in the first amplification system or the second amplification system, the concentration of the primer composition is 0.1-2.0 μM, the concentration of dNTPs is 1.0-2.0 mM, MgCl2 The concentration is 5 ~ 10mM.
The detection method according to claim 12, wherein the molar ratio of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP, the loop primer LF and the loop primer LB of the first primer set is 1: 1:8:8:4:4, the molar ratio of the outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, and loop primer LB of the second primer set is 1:1:8:8:4 .
The detection method according to claim 12, wherein the template of the first amplification system is an armored virus containing the Orflab gene target fragment, and the template of the second amplification system is an armored virus containing the Orflab gene target fragment. Virus.