CN113215312A

CN113215312A - Coronavirus SARS-CoV-2 digital PCR multiple detection kit and its application

Info

Publication number: CN113215312A
Application number: CN202110464355.6A
Authority: CN
Inventors: 李登旺; 陈振; 欧兰香; 蔡克海; 张绍明; 吴冰; 朱成龙; 丁兴龙; 李文靖
Original assignee: SHANDONG LAIBO BIOTECHNOLOGY CO Ltd
Current assignee: SHANDONG LAIBO BIOTECHNOLOGY CO Ltd
Priority date: 2021-04-28
Filing date: 2021-04-28
Publication date: 2021-08-06

Abstract

The invention discloses a coronavirus SARS-CoV-2 digital PCR multiple detection kit, which comprises a digital PCR reaction solution, a digital PCR primer mixed solution and a negative control solution, and is combined with a full-automatic micro-droplet chip digital PCR system to complete the detection of coronavirus SARS-CoV-2. The invention designs specific primer probes aiming at 3 target genes of coronavirus SARS-CoV-2 non-mutant ORF1ab gene, N gene and E gene and 4 mutant sites of N501Y and HV69-70del in B.1.1.7S gene of mutant strain thereof and E484K and K417N sites in B.1.351S gene of south Africa mutant strain, namely two mutant strains, improves the detection accuracy by detecting different sites of multiple genes, and the digital PCR calculates the target copy number by Poisson distribution, realizes single molecule detection and can quickly realize accurate judgment. The product of the invention enables the novel coronavirus digital PCR detection technology to better meet clinical requirements and be widely applied.

Description

Coronavirus SARS-CoV-2 digital PCR multiple detection kit and its application

Technical Field

The invention relates to a virus detection kit and application thereof, in particular to a coronavirus SARS-CoV-2 digital PCR multiple detection kit and application thereof, belonging to the technical field of clinical examination.

Background

The novel coronavirus, "SARS-CoV-2", is a new strain of coronavirus that has not been previously discovered in humans. After people are infected with coronavirus, the common signs of the person are respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death.

The nucleic acid detection is used as the gold standard of the current novel coronavirus detection method, and plays a vital role in the processes of rapid diagnosis, curative effect evaluation and epidemic prevention and control. However, the existing SARS-CoV-2 real-time fluorescence RT-PCR kit developed by various manufacturers rapidly is unstable, and especially after more than 5 detections are carried out on individual cases and convalescent patients at different time, the result is changed from negative to positive, which brings great difficulty for clinical diagnosis and disease control. Therefore, the real-time fluorescence RT-PCR still has certain application limitation on the detection of limited sample materials and early low-virus-content samples, for example, the detection process depends on the threshold (CT) of an amplification curve for quantification and is influenced by the amplification efficiency. In addition, in situations where new coronavirus is still abusive worldwide, variation of virus strains increasingly increases the difficulty of epidemic prevention and control. It has been reported that the propagation speed of UK novel coronavirus variant B.1.1.7 is 70% higher than that of the previous strain, and the lethality is 30% higher than that of the original virus; a new coronavirus strain b.1.351 found in south africa is believed to be at least 50% more infectious than the original virus, and more fearful is that the key mutations present in its spike protein render it resistant to vaccine or natural immunity. Since 1 month in 2021, the centers for controlling diseases in Shanghai, Guangdong, Shandong and the like in China also report successively, and the cases of infection of the new coronavirus B.1.1.7 mutant strain reported in England and the new coronavirus B.1.351 mutant strain reported in south Africa are found.

The Digital PCR (Digital PCR-dPCR) technology is a third-generation PCR technology, is a nucleic acid quantitative analysis technology, has simple operation, controllable quality and high sensitivity, and is the leading technology and the development direction in the PCR field. The digital PCR utilizes Poisson distribution to calculate the target copy number, realizes single molecule detection, and compared with qRT-PCR, the detection process does not depend on the threshold (CT) of an amplification curve for quantification, is not influenced by the amplification efficiency, and is simple and convenient to operate and high in sensitivity. It has wide application prospect in the fields of tumor liquid biopsy, companion diagnosis, genetic disease diagnosis, noninvasive prenatal examination, microorganism, infectious disease, food safety detection and the like.

Based on this, in order to better identify the new coronavirus species and effectively help the epidemic situation prevention and control, it is urgent and important to develop a multiple nucleic acid detection kit which is based on the digital PCR technology and can simultaneously detect the novel coronavirus non-mutant strain, the novel coronavirus mutant strain B.1.1.7 and the novel coronavirus south Africa mutant strain B.1.351.

Through retrieval, a novel coronavirus 7-channel digital PCR detection kit for simultaneously detecting novel coronavirus non-mutant strains, novel coronavirus mutant strains B.1.1.7 and novel coronavirus south Africa mutant strains B.1.351 by using 7 groups of specific probe primers is not reported.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a coronavirus SARS-CoV-2 digital PCR multiplex detection kit and application thereof.

The invention relates to a coronavirus SARS-CoV-2 digital PCR multiple detection kit, which comprises a digital PCR reaction solution, a digital PCR primer mixed solution and a negative control solution, and is combined with a full-automatic micro-droplet chip digital PCR system to complete the detection of coronavirus SARS-CoV-2; the digital PCR primer mixture liquid is a mixture liquid of a forward primer, a reverse primer and a primer probe for detecting the ORF1ab gene, the N gene, the E gene, the N501Y and the HV69-70del sites in the mutant strain B.1.1.7S gene and the E484K and K417N sites in the south Africa mutant strain B.1.351S gene, wherein the primer probe is provided with at least one fluorescent group;

the method is characterized in that:

the components of the digital PCR reaction solution are 250mM PCR buffer solution, which is pH8.0, Tris-HCl buffer solution and 20mM Mg²⁺Solution, 1mM of each of 4 dNTPs, 5U of c-MMLV enzyme, 10U of hot start Taq enzyme, 40U of RNase;

the forward primer, the reverse primer and the primer probe for detecting the coronavirus SARS-CoV-2 non-mutant ORF1ab gene are ORF1ab-F, ORF1ab-R and ORF1ab-P respectively; wherein the nucleotide sequence of the forward primer ORF1ab-F is 5'-GAGATGCCACAACTGCTT-3', the nucleotide sequence of the reverse primer ORF1ab-R is 5'-GCTATTCATACAGGCGTT-3', and the nucleotide sequence of the primer probe ORF1ab-P is 5 '-CY 5-AGTGTTTTTAACATTTGTCA-BHQ 3-3', wherein CY5 is a fluorescent group, and BHQ3 is a quenching group;

the forward primer, the reverse primer and the primer probe for detecting the N gene of the SARS-CoV-2 non-mutant strain of coronavirus are respectively N-F, N-R, N-P; the nucleotide sequence of the forward primer N-F is 5'-TGGACTTCCCTATGGTGC-3', the nucleotide sequence of the reverse primer N-R is 5'-AGCACGATGTTGAAGGAGT-3', and the nucleotide sequence of the primer probe N-P is 5 '-CY 5.5-CGGCATCATATGGGTTGCAA-BHQ 3-3', wherein CY5.5 is a fluorescent group, and BHQ3 is a quenching group;

the forward primer, the reverse primer and the primer probe for detecting the E gene of the SARS-CoV-2 non-mutant strain of the coronavirus are respectively E-F, E-R, E-P; the nucleotide sequence of the forward primer E-F is 5'-ATGTACTCATTCGTTTCG-3', the nucleotide sequence of the reverse primer E-R is 5'-CAATCGAAGCGCAGTAAG-3', and the nucleotide sequence of the primer probe E-P is 5 '-VIC-ATAGTTAATAGCGTACTTCT-BHQ 1-3', wherein VIC is a fluorescent group, and BHQ1 is a quenching group;

the forward primer, the reverse primer and the primer probe for detecting the N501Y site in the B.1.1.7S gene of the coronavirus SARS-CoV-2 mutant strain are respectively N501Y-F, N501Y-R, N501Y-P; the nucleotide sequence of the forward primer N501Y-F is 5'-CAATTTTGTAATGATCCA-3', the nucleotide sequence of the reverse primer N501Y-R is 5'-TGCAATTATTCGCACTAG-3', and the nucleotide sequence of the primer probe N501Y-P is 5 '-ROX-TGTTTACCACAAAAACAACA-MGB-3', wherein ROX is a fluorescent group, and MGB is a quenching group;

the forward primer, the reverse primer and the primer probe for detecting HV69-70del site in the B.1.1.7S gene of the coronavirus SARS-CoV-2 mutant strain are HV69-70del-F, HV69-70del-R, HV69-70del-P respectively; wherein the nucleotide sequence of the forward primer HV69-70del-F is 5'-CTTTTCCAATGTTACTT-3', the nucleotide sequence of the reverse primer HV69-70del-R is 5'-AATAAACACCATCATTAA-3', and the nucleotide sequence of the primer probe HV69-70del-P is 5 '-CY 7-TGCTATCTCTGGGACCAATGG-MGB-3', wherein CY7 is a fluorescent group and MGB is a quenching group;

the forward primer, the reverse primer and the primer probe for detecting the E484K site in the coronavirus SARS-CoV-2 south Africa mutant strain B.1.351S gene are respectively E484K-F, E484K-R, E484K-P; wherein the nucleotide sequence of the forward primer E484K-F is 5'-TCTATCAGGCCGGTAGC-3', the nucleotide sequence of the reverse primer E484K-R is 5'-CCATTAGTGGGTTGGAAA-3', and the nucleotide sequence of the primer probe E484K-P is 5 '-FAM-AATGGTGTTAAAGGTTTTAA-MGB-3', wherein FAM is a fluorescent group, and MGB is a quenching group;

the forward primer, the reverse primer and the primer probe for detecting the K417N site in the coronavirus SARS-CoV-2 south Africa mutant strain B.1.351S gene are respectively K417N-F, K417N-R, K417N-P; wherein the nucleotide sequence of the forward primer K417N-F is 5'-GATGAAGTCAGACAAATC-3', the nucleotide sequence of the reverse primer K417N-R is 5'-TTCCAAGCTATAACGCAG-3', and the nucleotide sequence of the primer probe K417N-P is 5 '-Atto 425-CTGGAAATATTGCTGATTAT-MGB-3', wherein Atto425 is a fluorescent group, and MGB is a quenching group;

the concentration of the forward primer or the reverse primer is 500-1000nM, and the concentration of the primer probe is 150-250 nM;

the negative control solution is non-ribozyme water treated by diethyl cokenate;

the total volume of the PCR reaction system of the coronavirus SARS-CoV-2 digital PCR multiple detection kit is 15 muL, wherein the adding amount of the digital PCR reaction solution is 3 muL, the adding amount of the digital PCR primer mixed solution is 2 muL, the adding amount of the nucleic acid sample is 3 muL, and the adding amount of the ribozyme-free water is 7 muL.

In the coronavirus SARS-CoV-2 digital PCR multiplex detection kit: the concentration of the forward primer or the reverse primer is preferably 500-700nM, and the concentration of the primer probe is preferably 150-200 nM.

The invention discloses an application of a coronavirus SARS-CoV-2 digital PCR multiplex detection kit in biological samples for detecting N501Y and HV69-70del sites in a gene containing a coronavirus SARS-CoV-2 non-mutant strain ORF1ab gene, an N gene and an E gene, a gene containing a coronavirus SARS-CoV-2 mutant strain B.1.1.7S gene and a biological sample containing a coronavirus SARS-CoV-2 south African mutant strain B.1.351S gene, E484K and K417N sites.

The method for detecting the sample comprises the following steps:

(1) extracting a nucleic acid sample: taking at least one of sputum, pharyngeal swab and alveolar lavage fluid of a detected person as a sample, and extracting RNA nucleic acid by using a commercially available RNA extraction kit according to the instruction of the kit;

(2) configuring a PCR reaction system; the total volume of the PCR reaction system is 15 mu L, wherein the adding amount of the digital PCR reaction solution is 3 mu L, the adding amount of the digital PCR primer mixed solution is 2 mu L, the adding amount of the nucleic acid sample is 3 mu L, and the adding amount of the ribozyme-free water is 7 mu L;

(3) generating liquid drops: transferring the reaction solution of the PCR reaction system into a droplet chip, sealing an oil phase, and putting the droplet chip into a sample preparation instrument for droplet preparation to generate droplets on the droplet chip;

(4) and (3) PCR amplification: putting the droplet chip generated by the droplets into a full-automatic droplet chip digital PCR system for PCR amplification, wherein the PCR reaction program is as follows: 15min at 50 ℃; 15min at 95 ℃; reacting at 94 ℃ for 15s and 60 ℃ for 30s for 40 cycles; 10min at 25 ℃ and 5min at 50 ℃; storing at 25 deg.C;

(5) chip reading and analysis and judgment of the results: placing the liquid drop chip subjected to PCR amplification into a biochip reader, photographing the liquid drop chip by the biochip reader, positioning and calculating liquid drops according to software to obtain the positive copy number concentration of each channel, and judging the negative and positive of the nucleic acid sample of the detected sample according to the positive copy number concentration of each channel; the reference standards are:

ORF1ab gene: if CY5 is more than or equal to 0.2 copy/mu L, the channel is judged to be positive; if CY5 is more than or equal to 0.1 copy/mu L and CY5 is less than 0.2 copy/mu L, the gray area is determined, the channel reports suspected positive, and the detection needs to be carried out again; if the CY5 is tested again and is less than 0.1 copy/mu L, the channel is negative or lower than the lower detection limit of the kit, and if the CY5 is more than or equal to 0.1 copy/mu L, the channel is reported to be positive;

e gene: if the VIC is more than or equal to 0.2 copy/mu L, the channel is judged to be positive; if the VIC is more than or equal to 0.1 copy/mu L and the VIC is less than 0.2 copy/mu L, the area is a gray area and needs to be detected again; if the repeated detection VIC is less than 0.1 copy/muL, the channel is negative or lower than the detection lower limit of the kit, and if the VIC is more than or equal to 0.1 copy/muL, the channel is reported to be positive;

n gene: if CY5.5 is more than or equal to 0.2 copy/mu L, the channel is judged to be positive; if CY5.5 is more than or equal to 0.1 copy/mu L and CY5.5 is less than 0.2 copy/mu L, the gray area is determined and needs to be detected again; if the CY5.5 is rechecked to be less than 0.1 copy/mu L, the channel is negative or lower than the lower detection limit of the kit, and if the CY5.5 is more than or equal to 0.1 copy/mu L, the channel is reported to be positive;

position N501Y: if ROX is more than or equal to 0.2 copy/mu L, the channel is judged to be positive; if ROX is more than or equal to 0.1 copy/mu L and ROX is less than 0.2 copy/mu L, the region is gray and needs to be detected again; if the retest ROX is less than 0.1 copy/mu L, the channel is negative or lower than the detection lower limit of the kit, and if the ROX is more than or equal to 0.1 copy/mu L, the channel is reported to be positive;

HV69-70del site: if CY7 is more than or equal to 0.2 copy/mu L, the channel is judged to be positive; if CY7 is more than or equal to 0.1 copy/mu L and CY7 is less than 0.2 copy/mu L, the gray area is determined and needs to be detected again; if the CY7 is tested again and is less than 0.1 copy/mu L, the channel is negative or lower than the lower detection limit of the kit, and if the CY7 is more than or equal to 0.1 copy/mu L, the channel is reported to be positive;

E484K site: FAM is more than or equal to 0.2 copy/mu L, and the channel is judged to be positive; if FAM is more than or equal to 0.1 copy/muL and FAM is less than 0.2 copy/muL, the gray area is determined and needs to be detected again; if the repeated detection FAM is less than 0.1 copy/muL, the channel is negative or lower than the detection lower limit of the kit, and if the FAM is more than or equal to 0.1 copy/muL, the channel is reported to be positive;

K417N site: if Atto425 is more than or equal to 0.2 copy/mu L, the channel is judged to be positive; if Atto425 is more than or equal to 0.1 copy/muL and CY5 is less than 0.2 copy/muL, the channel is a gray area, the channel reports suspected positive, and the detection needs to be carried out again; if the retest Atto425 is less than 0.1 copy/mu L, the channel is negative or lower than the detection lower limit of the kit, and if the Atto425 is more than or equal to 0.1 copy/mu L, the channel is reported to be positive;

wherein, if at least 2 channels in 3 channels of the ORF1ab gene, the N gene and the E gene are positive, the result of the nucleic acid sample is judged to be positive by the SARS-CoV-2 non-mutant strain of the coronavirus; if only 1 channel is positive, the result of the nucleic acid sample is judged to be positive by a suspected coronavirus SARS-CoV-2 non-mutant strain; if the 7 channels are negative, the result of the nucleic acid sample is judged to be negative to the coronavirus SARS-CoV-2; if the N501Y and HV69-70del site 2 channels in the sample which is judged to be positive are positive, the result of the nucleic acid sample is judged to be positive by the coronavirus SARS-CoV-2 mutant strain B.1.1.7S; if only 1 channel is positive, the nucleic acid sample result is judged to be positive by a suspected coronavirus SARS-CoV-2 mutant strain B.1.1.7S; if the E484K and K417N site 2 channels in the sample which is positive in the above interpretation are positive, the result of the nucleic acid sample is judged to be positive by the coronavirus SARS-CoV-2 south Africa mutant strain B.1.351; if only 1 channel is positive, the result of the nucleic acid sample is judged to be positive by the suspected coronavirus SARS-CoV-2 south Africa mutant strain B.1.351.

The invention discloses a coronavirus SARS-CoV-2 digital PCR multiple detection kit and its application, which is designed specific primer probes aiming at coronavirus SARS-CoV-2 non-mutant ORF1ab gene, N gene, 3 target genes of E gene and 4 mutant sites of N501Y, HV69-70del site in B.1.1.7S gene and E484K, K417N site in south African mutant strain B.1.351S gene, namely two mutant strains, the detection accuracy is improved by multi-gene different site detection, the digital PCR utilizes Poisson distribution to calculate target copy number, single molecule detection is realized, and accurate detection of novel coronavirus non-mutant strain, novel coronavirus mutant strain B.1.1.7 and novel coronavirus south African mutant strain B.1.351 can be rapidly realized. Compared with qRT-PCR, the detection process of the invention does not depend on the threshold (CT) of an amplification curve for quantification, is not influenced by the amplification efficiency, and has simple operation and high sensitivity. Meanwhile, the invention can simultaneously detect the novel coronavirus non-mutant strain, the novel coronavirus mutant strain B.1.1.7 and the novel coronavirus south Africa mutant strain B.1.351 by utilizing 7 groups of specific probe primers, so that the novel coronavirus digital PCR detection technology is more in line with clinical requirements and is more widely applied. The simultaneous detection of multiple targets can reduce the sample usage, shorten the detection time, save the reagent material, increase the detection flux and reduce the cost. According to the search, no report related to a novel coronavirus 7-channel digital PCR detection kit exists at present.

Drawings

FIG. 1 is a one-dimensional amplification chart of ORF1ab gene.

FIG. 2 is a one-dimensional amplification chart of the N gene.

FIG. 3 is a one-dimensional amplification chart of the E gene.

FIG. 4 is a one-dimensional amplification plot of site N501Y.

FIG. 5 is a one-dimensional amplification plot of the HV69-70del sites.

FIG. 6 is a one-dimensional amplification chart of E484K site.

FIG. 7 is a one-dimensional amplification plot of the K417N site.

Detailed Description

The present invention will be described in detail with reference to the following detailed drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention. Furthermore, it will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, and/or combinations thereof.

In the following examples, materials, reagents and the like used were obtained commercially unless otherwise specified.

Example 1: coronavirus SARS-CoV-2 digital PCR multiple detection reagent kit

The kit comprises a digital PCR reaction solution, a digital PCR primer mixed solution and a negative control solution, and is combined with a full-automatic micro-droplet chip digital PCR system to complete the detection of the coronavirus SARS-CoV-2.

Wherein:

the components of the digital PCR reaction solution are 250mM PCR buffer solution, which is pH8.0, Tris-HCl buffer solution and 20mM Mg²⁺Solution, 1mM of each of 4 dNTPs, 5U of c-MMLV enzyme, 10U of hot start Taq enzyme, 40U of RNase; the recipe of the digital PCR reaction solution is shown in Table 1.

Table 1: digital PCR reaction solution formula

The digital PCR primer mixed solution is a mixed solution of forward primers, reverse primers and primer probes for detecting the ORF1ab gene, the N gene, the E gene of a non-mutant strain of SARS-CoV-2, the N501Y and the HV69-70del sites in a mutant strain B.1.1.7S gene and the E484K and K417N sites in a south Africa mutant strain B.1.351S gene, wherein at least one fluorescent group is arranged on each primer probe; the specific digital PCR primers are:

the nucleotide sequences of the primers and the primer probes are shown in Table 2.

Table 2: primer and primer probe nucleotide sequence

The concentration formula of the forward primer or the reverse primer and the primer probe in the digital PCR primer mixture is shown in Table 3.

Table 3: formula of digital PCR primer mixed solution

the total volume of the PCR reaction system of the coronavirus SARS-CoV-2 digital PCR multiple detection kit is 15 muL, wherein the adding amount of the digital PCR reaction solution is 3 muL, the adding amount of the digital PCR primer mixed solution (SEQ ID NO: 1-SEQ ID NO: 21) is 2 muL, the adding amount of the nucleic acid sample is 3 muL, and the adding amount of the ribozyme-free water is 7 muL.

The formula of the PCR reaction system of the kit is shown in Table 4.

Table 4: kit PCR reaction system formula

Example 2:

the application of coronavirus SARS-CoV-2 digital PCR multiplex detection kit in detecting biological samples containing coronavirus SARS-CoV-2 non-mutant ORF1ab gene, N gene and E gene, N501Y and HV69-70del sites in coronavirus SARS-CoV-2 mutant strain B.1.1.7S gene and E484K and K417N sites in coronavirus SARS-CoV-2 south African mutant strain B.1.351S gene, wherein the related method for detecting samples is as follows:

(2) configuring a PCR reaction system; the total volume of the PCR reaction system is 15 mu L, wherein the adding amount of the digital PCR reaction solution is 3 mu L, the adding amount of the digital PCR primer mixture (SEQ ID NO: 1-SEQ ID NO: 21) is 2 mu L, the adding amount of the nucleic acid sample is 3 mu L, and the adding amount of the ribozyme-free water is 7 mu L;

Example 3: nucleic acid sample detection by full-automatic micro-droplet chip digital PCR instrument produced by Shandongbao biological science and technology limited

(1) Extracting a nucleic acid sample: taking at least one of sputum, pharyngeal swab and alveolar lavage fluid of a subject as a sample, and extracting RNA nucleic acid by using a commercially available RNA extraction Kit (such as QIAamp Viral RNA Mini Kit from Qiagen) according to the instruction of the Kit;

(3) opening a packaging box in a clean bench or a biological safety cabinet, taking out a liquid drop chip, and carefully tearing off an outer package of a chip bracket and a sealing film above a chip cup, wherein the chip is checked before use, if bubbles exist or the liquid level in the cup is obviously higher, a chip channel is unavailable, and the liquid drop chip needs to be placed on the chip bracket in the using process and has correct placement direction;

(4) slowly adding 15 μ L of PCR reaction system reaction solution into the inlet along the tube wall, covering the inlet and outlet with silica gel cap after PCR reaction solution gravity flows into the position below 20 μ L of oil phase liquid level to complete oil phase sealing, wherein when covering the silica gel cap, the silica gel cap is suspended tightly, and the upper vent hole is not pressed;

(5) placing the droplet chip and the bracket in a full-automatic droplet chip digital PCR instrument, selecting 'droplet generation', 'PCR amplification' and 'chip reading' on a software interface, and pressing a 'start button' to generate droplets;

(6) and (3) PCR amplification: the liquid drop chip with the generated liquid drops automatically enters a stage of 'PCR amplification' and 'chip reading', wherein a PCR reaction program is set as follows: 15min at 50 ℃; 15min at 95 ℃; reacting at 94 ℃ for 15s and 60 ℃ for 30s for 40 cycles; 10min at 25 ℃ and 5min at 50 ℃; storing at 25 deg.C;

(8) chip reading and analysis and judgment of the results: placing the liquid drop chip subjected to PCR amplification into a biochip reader, photographing the liquid drop chip by the biochip reader, positioning and calculating liquid drops according to software to obtain the positive copy number concentration of each channel, and judging the negative and positive of the nucleic acid sample of the detected sample according to the positive copy number concentration of each channel; as described in example 2 with reference to the standard.

Example 4: clinical sample detection and comparison

Because of the particularity of the coronavirus SARS-CoV-2 pneumonia, the kit of the invention is used for collecting throat swabs for only 10 employees, and additionally selecting 1 example of a coronavirus SARS-CoV-2 nucleic acid positive sample, 1 example of a mutant strain B.1.1.7 sample and 1 example of a Nanfei mutant strain B.1.351 sample, and respectively carrying out the fluorescent RT-PCR detection and the microdrop digital PCR detection of SARS-CoV-2, wherein the SARS-CoV-2 fluorescent RT-PCR detection uses a product which is already approved by the national drug administration, and the microdrop digital PCR detection of SARS-CoV-2 uses the method of the invention.

The results of 10 throat swabs from the 10 employees according to the digital PCR detection process and the commercial RT-PCR kit detection method of the present invention are shown in Table 5.

Table 5: negative sample result statistics

Sample numbering	Results of RT-PCR	Numerical PCR concentration values (copies/. mu.L)	Results
				1	Negative of	0	Uniformity
2	Negative of	0	Uniformity
				3	Negative of	0	Uniformity
4	Negative of	0	Uniformity
				5	Negative of	0	Uniformity
6	Negative of	0	Uniformity
				7	Negative of	0	Uniformity
8	Negative of	0	Uniformity
				9	Negative of	0	Uniformity
10	Negative of	0	Uniformity

According to the digital PCR detection process and the commercial RT-PCR kit detection method process, the detection results of 1 sample of nucleic acid positive sample of SARS-CoV-2 coronavirus, 1 sample of mutant strain B.1.1.7 and 1 sample of south Africa mutant strain B.1.351 are shown in Table 6.

Table 6: detection result of coronavirus SARS-CoV-2 nucleic acid positive sample digital PCR detection kit

The detection results show that the primer probe has normal specificity, no non-specific amplification exists in a normal human, a coronavirus SARS-CoV-2 nucleic acid positive sample, a mutant strain B.1.1.7 sample and a south African mutant strain B.1.351 sample, and the kit has good specificity.

In conclusion, combined with the application of clinical detection technology, the microdroplet chip digital PCR technology can be used for absolute nucleic acid quantification of novel coronavirus (SARS-CoV-2) with high sensitivity and high specificity in the true sense, and can be used for auxiliary diagnosis and virus load analysis of novel coronavirus (SARS-CoV-2) infection in the clinical field, in particular for detection of mutant strains. The kit has wide clinical application value and can help to prevent and control new crown epidemic situation.

Claims

1. A coronavirus SARS-CoV-2 digital PCR multiple detection kit comprises a digital PCR reaction solution, a digital PCR primer mixed solution and a negative control solution, and is combined with a full-automatic micro-droplet chip digital PCR system to complete the detection of coronavirus SARS-CoV-2; the digital PCR primer mixture liquid is a mixture liquid of a forward primer, a reverse primer and a primer probe for detecting the ORF1ab gene, the N gene, the E gene, the N501Y and the HV69-70del sites in the mutant strain B.1.1.7S gene and the E484K and K417N sites in the south Africa mutant strain B.1.351S gene, wherein the primer probe is provided with at least one fluorescent group;

the method is characterized in that:

the components of the digital PCR reaction solution are 250mM PCR buffer solution, which is pH8.0, Tris-HCl buffer solution and 20mM Mg²⁺Solution, 1mM each of 4 dNTPs, 5U of c-MMLV enzyme, 10U hot start Taq enzyme, 40U RNasin;

2. The digital PCR multiplex assay kit for SARS-CoV-2 coronavirus according to claim 1, wherein: the concentration of the forward primer or the reverse primer is 500-700nM, and the concentration of the primer probe is 150-200 nM.

3. Use of the coronavirus SARS-CoV-2 digital PCR multiplex assay kit as defined in claim 1 in the assay of biological samples containing coronavirus SARS-CoV-2 non-mutant ORF1ab gene, N gene and E gene, N501Y and HV69-70del in gene of coronavirus SARS-CoV-2 mutant strain B.1.1.7S, and E484K and K417N in gene of coronavirus SARS-CoV-2 south African mutant strain B.1.351S.

4. Use according to claim 3, wherein the method of detecting the sample is: