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

Abstract

The invention belongs to the field of biotechnology, and particularly relates to a qRT-PCR method for identifying Indian variant of novel coronavirus. 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 and a half of primers, that is, two upstream primers respectively target a mutation site and an original non-mutation site, and one downstream primer is shared by the two upstream primers. in order to improve the sensitivity of mutation detection, 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 virus nucleic acid amplified by mutation primer in the reaction system is earlier than the amplification curve of the non-mutation nucleic acid, and in the same way, the amplification curve of the non-mutation nucleic acid amplified by the non-mutation primer is earlier than the amplification curve of the mutated nucleic acid.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a qRT-PCR method for identifying the Indian variant of novel coronavirus. Background technique [0002] Gene sequencing is currently the most commonly used technology for identifying new coronavirus variants. This technology has the following disadvantages [1] : 1. The speed is slow 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 costs. The cost of novel ...

AI Technical Summary

Problems solved by technology

[0002] Gene sequencing is currently the most commonly used technology for identifying new coronavirus variants. This technology has the following disadvantages [1] : 1. Slow speed, many uncontrollable 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

The cost of novel coronavirus gene sequencing includes cumbersome 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

[0003] The above shortcomings of gene sequencing are applied to the prevention and control of the new crown pneumonia epidemic, which will greatly affect the timeliness, throughput and cost of variant detection, and may affect the detection results, directly affecting the prevention and control of the new crown pneumonia epidemic [2]

Images