CN113215313A - Detection kit for coronavirus SARS-CoV-2 and mutant strain thereof and application thereof - Google Patents

Detection kit for coronavirus SARS-CoV-2 and mutant strain thereof and application thereof Download PDF

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CN113215313A
CN113215313A CN202110464403.1A CN202110464403A CN113215313A CN 113215313 A CN113215313 A CN 113215313A CN 202110464403 A CN202110464403 A CN 202110464403A CN 113215313 A CN113215313 A CN 113215313A
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陈振
欧兰香
朱成龙
寇宗阳
王岩
贾秀玲
苏真真
高丽鹤
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SHANDONG LAIBO BIOTECHNOLOGY CO Ltd
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Abstract

The invention discloses a detection kit for coronavirus SARS-CoV-2 and its mutant strain, which comprises digital PCR reaction solution, digital PCR primer mixed solution and negative control solution, and combines with full-automatic micro-droplet chip digital PCR system to complete the detection of coronavirus SARS-CoV-2 and its mutant strain. The invention designs specific primer probes aiming at 3 mutant sites of E484K, L452R and D614G sites in 2 target genes and mutant strains S genes of coronavirus SARS-CoV-2 non-mutant ORF1ab genes and N genes, improves the detection accuracy of the kit through the digital PCR detection of different sites of polygenes, and realizes the rapid judgment. The kit provided by the invention better meets clinical requirements in novel coronavirus detection, screening and vaccine development, and also enables detection to be faster and wider in application.

Description

Detection kit for coronavirus SARS-CoV-2 and mutant strain thereof and application thereof
Technical Field
The invention relates to a virus detection kit and application thereof, in particular to a digital PCR detection kit for coronavirus SARS-CoV-2 and mutant strains thereof 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.
Close tracking of new coronavirus mutants is now a focus of research by scientists worldwide, and the study of these mutations is useful in analyzing viral transmission and immune resistance. The new crown epidemic has lasted for one year, and from the current data, the new crown virus has not changed rapidly, about half of the influenza virus, one quarter of the HIV virus, and about two bases per month in the RNA genome of about 3 ten thousand bases of the new crown virus.
But 3 rapidly propagating mutants appeared recently with much attention. These 3 mutants were found in uk, south africa and brazil, respectively, and share the same genetic locus mutation E484K in addition to having more mutation sites. In the E484K mutation, the negatively charged amino acid (glutamic acid) was replaced by a positively charged amino acid (lysine), and structural analysis showed that a new ACE2 binding site was generated due to the E484K mutation. This may result in a significantly stronger interaction between ACE2 and the natural binding site located on the RBD. The E484K mutation makes the new coronavirus more contagious, and various studies have indicated that E484K also results in the ability of the new coronavirus to escape from immunity. Several studies have now shown that the E484K mutation may be critical in causing a reduction in vaccine protection efficacy. In india, a new coronavirus was found with both mutations E484Q and L452R. The university of Stanford clinical virology laboratory Master Pinsky (Benjamin Pinsky) showed that two key mutations, respectively, had been present in other variants, but have never been co-found in a single strain before. The L452R mutation will make the virus more transmissible, reducing the effectiveness of the vaccine. Meanwhile, laboratory studies find that the mutation of the new coronavirus D614G can lead to the accelerated replication and the enhancement of the transmissibility of the virus, and officials of world health organization indicate that: studies have shown that this variation occurs in 29% of the new coronavirus specimens, and viruses with this variation have been transmitted in europe and america.
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 the above data and results, it was shown that the novel coronavirus became more and more evasive in continuous mutation. The development of corresponding digital PCR detection kits aiming at the mutant strains aiming at the mutation-prone sites is accelerated, so that the prevention and control of the world complex epidemic situation in China are facilitated, and an effective barrier is established.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a digital PCR detection kit for coronavirus SARS-CoV-2 and mutant strains thereof and application thereof.
The invention relates to a detection kit for coronavirus SARS-CoV-2 and its mutant strain, which is a digital PCR multiple mutation detection kit, comprising digital PCR reaction solution, digital PCR primer mixed solution and negative control solution, and combining with full-automatic micro-droplet chip digital PCR system to complete the detection of coronavirus SARS-CoV-2 and its mutant strain; the digital PCR primer mixture is a mixture of a forward primer, a reverse primer and a primer probe for detecting the sites of E484K, L452R and D614G in the genes of SARS-CoV-2 non-mutant ORF1ab, N and mutant strain S, and 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 '-VIC-AGTGTTTTTAACATTTGTCA-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 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-CGGCATCATATGGGTTGCAA-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 E484K site in the S gene of the coronavirus SARS-CoV-2 mutant strain 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 L452R site in the S gene of the coronavirus SARS-CoV-2 mutant strain are respectively L452R-F, L452R-R, L452R-P; wherein the nucleotide sequence of the forward primer L452R-F is 5'-CTTGATTCTAAGGTTGGT-3', the nucleotide sequence of the reverse primer L452R-R is 5'-ACACCATTACAAGGTGTG-3', and the nucleotide sequence of the primer probe L452R-P is 5 '-ROX-TAATTACCGGTATAGATTGT-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 the D614G site in the S gene of the coronavirus SARS-CoV-2 mutant strain are respectively D614G-F, D614G-R, D614G-P; wherein the nucleotide sequence of the forward primer D614G-F is 5'-ATAACACCAGGAACAAATAC-3', the nucleotide sequence of the reverse primer D614G-R is 5'-ACGCCAAGTAGGAGTAAG-3', and the nucleotide sequence of the primer probe D614G-P is 5 '-Atto 425-ATCAGGGTGTTAACTGCACAGAA-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 detection kit for the coronavirus SARS-CoV-2 and the mutant strain thereof 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.
In the detection kit for the coronavirus SARS-CoV-2 and the mutant strain thereof: 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 detection kit for coronavirus SARS-CoV-2 and mutant strains thereof in detecting biological samples containing coronavirus SARS-CoV-2 non-mutant ORF1ab gene, N gene and coronavirus SARS-CoV-2 mutant strain S gene at E484K, L452R and D614G 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 at 59 ℃ 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 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 channel is a gray area, the channel reports suspected positive, and the detection needs to be carried out 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 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 and needs to be detected 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;
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;
L452R site: 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;
D614G 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 2 channels in the ORF1ab gene and the N gene 2 channel are positive, the result of the nucleic acid sample is judged to be positive by the coronavirus SARS-CoV-2 non-mutant strain; 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 5 channels are negative, the result of the nucleic acid sample is judged to be negative to the coronavirus SARS-CoV-2; if any channel of E484K, L452R and D614G site 3 channels in the sample judged to be positive by the above interpretation is positive, the result of the nucleic acid sample is judged to be positive by the coronavirus SARS-CoV-2 mutant strain.
The invention discloses a digital PCR detection kit for coronavirus SARS-CoV-2 and mutant strain thereof and application thereof, which is characterized in that specific primer probes are designed aiming at 3 mutant sites of E484K, L452R and D614G in SARS-CoV-2 non-mutant ORF1ab gene, 2 target genes of N gene and mutant strain S gene thereof, the detection accuracy is improved by multi-gene different site detection, the digital PCR calculates the target copy number by Poisson distribution, single molecule detection is realized, and the accurate detection of novel coronavirus non-mutant strain and mutant strain thereof can be quickly 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 and the mutant strain thereof by utilizing 5 groups of specific probe primers, can effectively distinguish which mutant strain exists in a human body, and combines a digital PCR technology, so that the kit disclosed by the invention is more in line with clinical requirements during novel coronavirus detection, screening and vaccine development, and is also quicker in detection and wider in application. And the simultaneous detection of multiple targets can reduce the sample consumption, shorten the detection time, save reagent materials, increase the detection flux and reduce the cost, and has economic value and social benefit.
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 E484K site.
FIG. 4 is a one-dimensional amplification plot of site L452R.
FIG. 5 is a one-dimensional amplification plot of site D614G.
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: digital PCR detection kit for SARS-CoV-2 coronavirus and mutant strain thereof
The kit of the invention comprises: the detection of the coronavirus SARS-CoV-2 and its mutant strain is completed by combining the digital PCR reaction solution, the digital PCR primer mixed solution and the negative control solution with the full-automatic micro-droplet chip digital PCR system.
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
Figure BDA0003043116060000051
Figure BDA0003043116060000061
The digital PCR primer mixed solution is a mixed solution of a forward primer, a reverse primer and a primer probe for detecting the sites of E484K, L452R and D614G in SARS-CoV-2 non-mutant ORF1ab gene, N gene and mutant strain S gene, wherein the primer probe is provided with at least one fluorescent group; the specific digital PCR primers are:
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 '-VIC-AGTGTTTTTAACATTTGTCA-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 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-CGGCATCATATGGGTTGCAA-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 E484K site in the S gene of the coronavirus SARS-CoV-2 mutant strain 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 L452R site in the S gene of the coronavirus SARS-CoV-2 mutant strain are respectively L452R-F, L452R-R, L452R-P; wherein the nucleotide sequence of the forward primer L452R-F is 5'-CTTGATTCTAAGGTTGGT-3', the nucleotide sequence of the reverse primer L452R-R is 5'-ACACCATTACAAGGTGTG-3', and the nucleotide sequence of the primer probe L452R-P is 5 '-ROX-TAATTACCGGTATAGATTGT-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 the D614G site in the S gene of the coronavirus SARS-CoV-2 mutant strain are respectively D614G-F, D614G-R, D614G-P; wherein the nucleotide sequence of the forward primer D614G-F is 5'-ATAACACCAGGAACAAATAC-3', the nucleotide sequence of the reverse primer D614G-R is 5'-ACGCCAAGTAGGAGTAAG-3', and the nucleotide sequence of the primer probe D614G-P is 5 '-Atto 425-ATCAGGGTGTTAACTGCACAGAA-MGB-3', wherein Atto425 is a fluorescent group and MGB is a quenching group;
the nucleotide sequences of the primers and the primer probes are shown in Table 2.
Table 2: primer and primer probe nucleotide sequence
Figure BDA0003043116060000071
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
Figure BDA0003043116060000072
Figure BDA0003043116060000081
The negative control solution is non-ribozyme water treated by diethyl cokenate;
the total volume of the PCR reaction system of the digital PCR detection kit for the coronavirus SARS-CoV-2 and the mutant strain thereof is 15 mu L, wherein the addition amount of the digital PCR reaction solution is 3 mu L, the addition amount of the digital PCR primer mixed solution (SEQ ID NO: 1-SEQ ID NO: 15) is 2 mu L, the addition amount of the nucleic acid sample is 3 mu L, and the addition amount of the ribozyme-free water is 7 mu L.
The formula of the PCR reaction system of the kit is shown in Table 4.
Table 4: kit PCR reaction system formula
Figure BDA0003043116060000082
Example 2:
application of digital PCR detection kit of coronavirus SARS-CoV-2 and its mutant strain in detection of biological samples containing coronavirus SARS-CoV-2 non-mutant ORF1ab gene, N gene, E484K, L452R and D614G locus in S gene containing coronavirus SARS-CoV-2 mutant strain, wherein the related method for detecting samples is as follows:
(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 (SEQ ID NO: 1-SEQ ID NO: 15) 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 at 59 ℃ 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 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 channel is a gray area, the channel reports suspected positive, and the detection needs to be carried out 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 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 and needs to be detected 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;
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;
L452R site: 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;
D614G 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 2 channels in the ORF1ab gene and the N gene 2 channel are positive, the result of the nucleic acid sample is judged to be positive by the coronavirus SARS-CoV-2 non-mutant strain; 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 5 channels are negative, the result of the nucleic acid sample is judged to be negative to the coronavirus SARS-CoV-2; if any channel of E484K, L452R and D614G site 3 channels in the sample judged to be positive by the above interpretation is positive, the result of the nucleic acid sample is judged to be positive by the coronavirus SARS-CoV-2 mutant strain.
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;
(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) 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 at 59 ℃ 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 verification
Due to the particularity of the coronavirus SARS-CoV-2 pneumonia, the kit provided by the invention is used for carrying out digital PCR detection on a coronavirus SARS-CoV-2 micro-droplet chip by only collecting throat swabs of 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 south Africa mutant strain B.1.351 sample, wherein the method provided by the invention is used for the digital PCR detection of the coronavirus SARS-CoV-2 micro-droplet chip.
The results of 10 throat swabs from employees tested according to the digital PCR assay protocol of the present invention are shown in Table 5.
Table 5: negative sample result statistics
Sample numbering Numerical PCR concentration values (copies/. mu.L) Results
1 0 Negative of
2 0 Negative of
3 0 Negative of
4 0 Negative of
5 0 Negative of
6 0 Negative of
7 0 Negative of
8 0 Negative of
9 0 Negative of
10 0 Negative of
The results of 1 sample positive for nucleic acid of coronavirus SARS-CoV-2, 1 sample of mutant strain B.1.1.7, and 1 sample of mutant strain B.1.351 in south Africa according to the digital PCR detection process of the present invention are shown in Table 6.
Table 6: detection result of coronavirus SARS-CoV-2 nucleic acid positive sample digital PCR detection kit
Figure BDA0003043116060000111
Figure BDA0003043116060000121
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 (4)

1. A detection kit for coronavirus SARS-CoV-2 and its mutant strain is a digital PCR multiple mutation detection kit, which comprises digital PCR reaction solution, digital PCR primer mixed solution and negative control solution, and combines with full-automatic micro-droplet chip digital PCR system to complete the detection of coronavirus SARS-CoV-2 and its mutant strain; the digital PCR primer mixture is a mixture of a forward primer, a reverse primer and a primer probe for detecting the sites of E484K, L452R and D614G in the genes of SARS-CoV-2 non-mutant ORF1ab, N and mutant strain S, and 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 '-VIC-AGTGTTTTTAACATTTGTCA-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 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-CGGCATCATATGGGTTGCAA-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 E484K site in the S gene of the coronavirus SARS-CoV-2 mutant strain 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 L452R site in the S gene of the coronavirus SARS-CoV-2 mutant strain are respectively L452R-F, L452R-R, L452R-P; wherein the nucleotide sequence of the forward primer L452R-F is 5'-CTTGATTCTAAGGTTGGT-3', the nucleotide sequence of the reverse primer L452R-R is 5'-ACACCATTACAAGGTGTG-3', and the nucleotide sequence of the primer probe L452R-P is 5 '-ROX-TAATTACCGGTATAGATTGT-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 the D614G site in the S gene of the coronavirus SARS-CoV-2 mutant strain are respectively D614G-F, D614G-R, D614G-P; wherein the nucleotide sequence of the forward primer D614G-F is 5'-ATAACACCAGGAACAAATAC-3', the nucleotide sequence of the reverse primer D614G-R is 5'-ACGCCAAGTAGGAGTAAG-3', and the nucleotide sequence of the primer probe D614G-P is 5 '-Atto 425-ATCAGGGTGTTAACTGCACAGAA-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 detection kit for the coronavirus SARS-CoV-2 and the mutant strain thereof 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.
2. The detection kit for coronavirus SARS-CoV-2 and its mutant strain 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. The use of the detection kit for coronavirus SARS-CoV-2 and its mutant strain as claimed in claim 1 in the detection of biological samples containing coronavirus SARS-CoV-2 non-mutant ORF1ab gene, N gene, and coronavirus SARS-CoV-2 mutant strain S gene at E484K, L452R and D614G sites.
4. Use according to claim 3, wherein the method of detecting the sample is:
(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 at 59 ℃ 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 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 channel is a gray area, the channel reports suspected positive, and the detection needs to be carried out 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 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 and needs to be detected 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;
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;
L452R site: 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;
D614G 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 2 channels in the ORF1ab gene and the N gene 2 channel are positive, the result of the nucleic acid sample is judged to be positive by the coronavirus SARS-CoV-2 non-mutant strain; 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 5 channels are negative, the result of the nucleic acid sample is judged to be negative to the coronavirus SARS-CoV-2; if any channel of E484K, L452R and D614G site 3 channels in the sample judged to be positive by the above interpretation is positive, the result of the nucleic acid sample is judged to be positive by the coronavirus SARS-CoV-2 mutant strain.
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