Composition, kit and method for detecting SARS-CoV-2 variant and application
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A sars-cov-2, sars-cov-2n technology, applied in the field of molecular biology detection, can solve problems such as reducing the protective efficacy of antibody drugs
Active Publication Date: 2022-01-28
SHENZHEN UNI MEDICA TECH
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The superposition of mutations may reduce the protective efficacy of some antibody drugs against
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Embodiment 1
[0077] Embodiment 1, primers and probes used in the present invention
[0078] Table 1
[0079]
[0080]
[0081] Wherein, the fluorescent reporter group of SEQ ID NO:3 and 21 is HEX; The fluorescent reporter group of SEQ ID NO:6 and 9 is ROX; The fluorescent reporter group of SEQ ID NO:12 is CY5; SEQ ID NO:15 and The fluorescent reporter group of 18 is FAM, and the 3' end of the probe also has a BHQ1 quencher group.
Embodiment 2
[0082] Embodiment 2, the method for detecting SARS-CoV-2
[0085] Use commercial RNA extraction kits, such as nucleic acid extraction reagents based on silica gel membrane spin column method or nucleic acid extraction reagents based on magnetic bead method, and operate according to the kit instructions, and finally collect 80 μL RNA solution for direct detection.
[0086] Or store at -80°C. Both negative quality control and positive quality control need to be extracted;
[0087] 3. System configuration:
[0088] According to the total reaction number N required for detection, add 18.5 μl RT-PCR amplification solution (MgCl 2 6mM, dNTP0.8mM, and upstream primer / downstream primer concentration is 0.2μM, probe concentration is 0.25μM, universal upstream primer 0.6μM, universal downstream primer 6μM) and 1.5 μl enzyme mixture (hot start Taq enzyme, reverse transcriptase, ...
Embodiment 3
[0100] Embodiment 3, the detection result of the composition test sample of the present invention
[0101] The primers and probes shown in Example 1 are detected according to the method of Example 2 to the pseudovirus of Omicron, and the experimental results are as follows figure 1 shown. The results show that all channels can be detected normally, and the multiplex PCR system can detect the corresponding target and type the new crown mutant strains to identify the Omicron mutant strains.
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Abstract
The invention belongs to the field of molecular biological detection, and relates to the detection of a SARS-CoV-2 Omicron variant. The invention provides a kit containing the composition, application of the composition and a method for detecting and typing the SARS-CoV-2 variant. By detecting four different characteristic function variation sites on the S gene of the SARS-CoV-2 variant, the Omicron variant is subjected to typing, so that the typing detection of the SARS-CoV-2 virus and the Omicron variant is simultaneously realized in a single-tube reaction system. The composition disclosed by the invention is combined with a fluorescent probe melting curve method, and is low in cost and high in flux. Moreover, the operation is simple and convenient, and the result reading process can be judged through a melting peak Tm value. The whole detection process is carried out under a single-tube closed condition, so that false positive and environmental pollution caused by crossing of samples are avoided.
Description
technical field [0001] The invention belongs to the field of molecular biology detection; specifically, it relates to the detection of SARS-CoV-2; more specifically, it relates to the detection of SARS-CoV-2 Omicron variant strain. Background technique [0002] Novel coronavirus (2019-nCoV, SARS-CoV-2), on February 11, 2020, the International Virus Taxonomy Committee named the virus as SARS-CoV-2. Coronavirus is a large family of viruses. Before that, 6 species were known to infect humans, such as colds, Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). SARS-CoV-2 It is a new strain of coronavirus that has never been found in humans before. The virus belongs to the β-type coronavirus in the family Coronaviridae (Coronavirus) in taxonomy, and has the characteristics of capsule and spike cytology The genome is a linear single-stranded positive-sense RNA ((+)ssRNA) virus. [0003] Common signs of COVID-19 include respiratory symptoms such a...
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