Composition, kit and method for detecting coronaviruses
A technology of coronavirus and composition, applied in biochemical equipment and methods, methods based on microorganisms, microorganisms, etc., can solve the problem of no specific treatment for diseases and achieve good stability
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Embodiment 1
[0035] The preparation of embodiment 1 kit
[0036] The preparation method of the kit is exemplified below.
[0037]The kit includes tube 1, which is a primer mixture for amplifying the novel coronavirus (SARS-CoV-2) ORF1ab and N genes. The primer mixture consists of 4 primer sequences, and the specific primer sequences are shown in Table 1. In one case, tube 1 also contains a probe mixture, which is respectively the probes of the novel coronavirus (SARS-CoV-2) ORF1ab and the N gene conserved region, respectively labeled with different fluorescent molecules, in this implementation In the example, the probe of ORF1ab gene marks FAM, and the N gene marks HEX. The specific sequences of the probes are shown in Table 1. Tube 1 of each primer and each probe was used in a concentration range of 8-15uM, preferably 10uM. The novel coronavirus (SARS-CoV-2) ORF1ab and N genes can be effectively amplified by using the kit containing tube 1, and the copy number of the novel coronavirus ...
Embodiment 2
[0084] Embodiment 2 specific detection
[0085] With influenza A virus H1N1 (pdm09), influenza A virus H3N2, influenza B virus Victoria, influenza B virus (Yamagata), influenza C virus, respiratory syncytial virus type A, respiratory syncytial virus type B, Human bocavirus, human parainfluenza virus, human metapneumovirus, rotavirus / enterovirus, adenovirus, human coronavirus (HKU1, OC43, 229E and NL63), Middle East respiratory syndrome coronavirus and other viral nucleic acids as templates, The fluorescent quantitative PCR detection was carried out using the above kit, and the reaction system configuration during detection was set according to Table 3, and the reaction process was set according to Table 4. The test results showed that the above virus test results were all negative.
Embodiment 3
[0086] Application of embodiment 3 kit
[0087] The kit prepared in Example 1 is used for detection of ORF1ab and / or N gene amplification and non-diagnostic purposes containing novel coronavirus (SARS-CoV-2), for example, whether ORF1ab and / or N gene are contained in the plasmid, pure Detection of virus sample concentration, detection of fake virus amount, etc.
[0088] During the detection process, calculate the total number of reactions (N) according to the number of samples to be tested, making sure to include the negative control (1 reaction), positive control (1 reaction) and each sample. According to the number of reactions (N), prepare the PCR reaction system according to Table 6.
[0089] Table 6
[0090]
[0091] In this example, the 2019-nCOV pseudovirus was used as the test sample. The pseudovirus was purchased from Shanghai Yisheng Biotechnology Co., Ltd., the article number is 11900ES03, and the 2019-nCOV pseudovirus is the integration of some genes of the COVI...
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