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

A coronavirus, a new type of technology, applied in biochemical equipment and methods, microbial measurement/inspection, and resistance to vector-borne diseases, etc., can solve the impact on the timeliness, throughput and cost of variant detection, and affect the prevention and control of the new crown pneumonia epidemic , detection of high material and labor costs, etc., to achieve the effect of easy high-throughput operation, reduced material and labor costs, and reduced chance of operational contamination

Pending Publication Date: 2021-08-13
SHANXI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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

Method used

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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] A qRT-PCR method for identifying the Indian variant of novel coronavirus, comprising the following steps:

[0038] Step 1, use the Trizol method to extract the RNA of the new coronavirus;

[0039] Step 2, ARMS-based qRT-PCR primer probe design:

[0040] Step 2.1, primer design: According to the general principles of primer design, combined with the nucleotide sequence near the mutation site of the new coronavirus Indian variant, two pairs of primers were designed for each mutation site, in which the 3' ends of the upstream primers matched the mutation and non-coronavirus respectively. Variation point, that is, a variation upstream primer and a non-variation upstream primer, two pairs of upstream primers share a downstream primer;

[0041] When the variable site to be identified is L452R, the nucleotide sequence of the mutated upstream primer is shown in SEQ ID NO.1: 5'-GATTCTAAGGTTGGTGGTAATTATAATTATCG-3', and the nucleotide sequence of the non-variant upstream primer i...

Embodiment 2

[0055] Method of the present invention is done sensitivity experiment (template dilution 10 6 -10 -1 copy / microliter), the experimental results are as follows figure 1 As shown, when the variable site to be identified is L452R, the detection sensitivity is 0.80 copies / microliter; when the variable site to be identified is T478K, the detection sensitivity is 2.1 copies / microliter; when the variable site to be identified is When it is P681R, the detection sensitivity is 3.3 copies / microliter.

Embodiment 3

[0057] The precision of the repeatability test evaluation method refers to the closeness between a series of single measured values ​​obtained by repeated measurements of the same specimen under certain conditions, and is an index reflecting the size of random errors. It is divided into intra-batch repeatability experiments, Inter-batch repeatability experiments (intra-day, day-to-day repeatability experiments), operator / instrument repeatability experiments, etc.

[0058] We investigated the repeatability (precision) of this method, measured by the coefficient of variation, and the experimental results are shown in Table 2.

[0059] Table 2. Repeatability (precision) of the method

[0060]

[0061] The calculation method of the coefficient of variation is:

[0062] Coefficient of variation (%) = (Ct standard deviation / Ct average) × 100%

[0063] It can be seen from Table 2 that the range of the intra-assay coefficient of variation of L452R is 0.20%-1.25%, and the range ...

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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 ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6858
CPCC12Q1/701C12Q1/6858C12Q2521/107C12Q2535/137C12Q2531/113C12Q2561/101Y02A50/30
Inventor 李建国高泽峰李珍夏娟吴长新
Owner SHANXI UNIV
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