Coronavirus SARS-CoV-2 digital PCR multiple detection kit and application thereof
A coronavirus, multiple detection technology, applied in the field of clinical testing, to shorten the detection time, increase the detection throughput, and have a wide range of applications.
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Embodiment 1
[0047] Embodiment 1: Coronavirus SARS-CoV-2 digital PCR multiple detection kit
[0048] The kit of the invention comprises a digital PCR reaction solution, a digital PCR primer mixture and a negative control solution, and is combined with a fully automatic microdrop chip digital PCR system to complete the detection of the coronavirus SARS-CoV-2.
[0049] in:
[0050] The components of the digital PCR reaction solution are 250mM PCR buffer, which is pH8.0, Tris-Hcl buffer, 20mM Mg 2+ Solution, 1 mM of each of the four dNTPs, 5 U of c-MMLV enzyme, 10 U of hot-start Taq enzyme, and 40 U of RNasin; the formula of the digital PCR reaction solution is shown in Table 1.
[0051] Table 1: Digital PCR Reaction Solution Recipe
[0052]
[0053] The digital PCR primer mixture is used to detect the N501Y, HV69-70del sites and South African mutations in the ORF1ab gene, N gene, E gene, mutant strain B.1.1.7S gene of the coronavirus SARS-CoV-2 non-mutant strain A mixture of forward pr...
Embodiment 2
[0073] The coronavirus SARS-CoV-2 digital PCR multiplex detection kit is used to detect the ORF1ab gene, N gene and E gene of the coronavirus SARS-CoV-2 non-mutant strain, and the coronavirus SARS-CoV-2 mutant strain B.1.1. The application of N501Y and HV69-70del sites in the 7S gene and E484K and K417N sites in the South African mutant strain of coronavirus SARS-CoV-2 B.1.351S gene to biological samples, the methods involved in the detection of samples are:
[0074] (1) Nucleic acid sample extraction: take at least one of the subject's sputum, throat swab, and alveolar lavage fluid as a sample, use a commercially available RNA extraction kit, and follow the kit's instructions to extract RNA nucleic acid;
[0075] (2) The configuration of the PCR reaction system; the total volume of the PCR reaction system is 15 μL, the amount of digital PCR reaction solution added is 3 μL, and the amount of digital PCR primer mixture (SEQ ID NO: 1~SEQ ID NO: 21) added is 2 μL, 3 μL for nuclei...
Embodiment 3
[0087]Example 3: Detection of nucleic acid samples using a fully automatic micro-droplet chip digital PCR instrument produced by Shandong Laibo Biotechnology Co., Ltd.
[0088] (1) Nucleic acid sample extraction: Take at least one of the subject’s sputum, throat swab, and alveolar lavage fluid as a sample, use a commercially available RNA extraction kit (such as Qiagen’s QIAamp Viral RNA Mini Kit), and follow the reagent The instructions of the box are used to extract RNA nucleic acid;
[0089] (2) The configuration of the PCR reaction system; the total volume of the PCR reaction system is 15 μL, in which the amount of digital PCR reaction solution added is 3 μL, the amount of digital PCR primer mixture added is 2 μL, the amount of nucleic acid sample added is 3 μL, nuclease-free The amount of water added is 7 μL;
[0090] (3) Open the packing box in the ultra-clean workbench or biological safety cabinet, take out the droplet chip, and carefully tear off the outer package of ...
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