QRT-PCR (quantitative reverse transcription-polymerase chain reaction) method for identifying novel coronavirus Gamma variant

Abstract

The invention belongs to the technical field of biology, and particularly relates to a qRT-PCR (quantitative reverse transcription-polymerase chain reaction) method for identifying a novel coronavirus Gamma variant. According to the method, in order to improve the detection sensitivity and specificity, a TaqMan probe is introduced; in order to reduce the cost, two pairs of primers are changed into one pair of half primers, that is, two upstream primers respectively target a mutation site and an original non-mutated site, and one downstream primer is shared by the two upstream primers. The sensitivity of variation detection is enhanced; mutation is introduced to the third site in the 5'direction of the mutation site of the upstream mutation primer, and by reducing the matching degree of the mutation primer and non-mutated virus nucleic acid and the matching degree of the non-mutated primer and the mutation virus nucleic acid in the reaction system, the amplification curve of the mutation primer for amplifying the mutation virus nucleic acid in the reaction system is earlier than the amplification curve of the non-mutated nucleic acid; the amplification curve of the non-variant nucleic acid amplified by the non-variant primer is earlier than the amplification curve of the variant nucleic acid amplified by the non-variant primer.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a qRT-PCR method for identifying a novel coronavirus Gamma mutant strain. Background technique [0002] Gene sequencing is currently the most commonly used technology for identification of new coronavirus variants. This technology has the following disadvantages [1]: 1. It takes a long time from sample to result, and there are many uncontrollable influencing factors. It takes 6-8 hours from sample processing to result reporting. In addition, most virus detection units do not have the conditions for gene sequencing and need to be tested by a third-party sequencing company. The speed is slow, and there are many uncontrollable influencing factors; 2. The detection sensitivity is low. Gene sequencing requires gene amplification before testing on the machine, and the slightly poor amplification effect on low-abundance nucleic acid samples can affect sequencing; 3. High detect...

AI Technical Summary

Problems solved by technology

[0002] Gene sequencing is currently the most commonly used technology for identification of new coronavirus variants. This technology has the following disadvantages [1]: 1. It takes a long time from sample to result, and there are many uncontrollable influencing factors

It takes 6-8 hours from sample processing to result reporting. In addition, most virus detection units do not have the conditions for gene sequencing and need to be tested by a third-party sequencing company. The speed is slow, and there are many uncontrollable influencing factors; 2. The detection sensitivity is low

Gene sequencing requires gene amplification before testing on the machine, and the slightly poor amplification effect on low-abundance nucleic acid samples can affect sequencing; 3. High detection cost

New coronavirus gene sequencing includes tedious steps such as sample nucleic acid extraction, RT-PCR, PCR product purification, and computer testing, resulting in high testing material and labor costs, and multi-step operations are prone to deviations in test results due to careless operations

The above shortcomings make gene sequencing when applied to the prevention and control of the new crown pneumonia epidemic, which will affect the detection timeliness, throughput and cost of virus variants, and may affect the detection results, which will have an important impact on the prevention and control of the new crown pneumonia epidemic [2]

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