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QRT-PCR (quantitative reverse transcription-polymerase chain reaction) method for identifying novel coronavirus Omicro variant BA-2 branch

A coronavirus, BA-2 technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem of affecting the detection time, throughput and cost of virus variant strains, and the high cost of detection materials and labor , many uncontrollable influencing factors, etc., to achieve the effect of easy high-throughput operation, reducing the probability of operation pollution, and improving the detection throughput

Pending Publication Date: 2022-06-24
SHANXI UNIV
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  • Summary
  • 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. 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]

Method used

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  • QRT-PCR (quantitative reverse transcription-polymerase chain reaction) method for identifying novel coronavirus Omicro variant BA-2 branch
  • QRT-PCR (quantitative reverse transcription-polymerase chain reaction) method for identifying novel coronavirus Omicro variant BA-2 branch
  • QRT-PCR (quantitative reverse transcription-polymerase chain reaction) method for identifying novel coronavirus Omicro variant BA-2 branch

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] A qRT-PCR method for identifying branches of novel coronavirus Omicron variant strain BA-2, comprising the following steps:

[0045] Step 1, use the TrizoL method to extract the new coronavirus RNA;

[0046] Step 2, ARMS-based qRT-PCR primer probe design:

[0047] Step 2.1, primer design: According to the general principles of primer design, two pairs of primers are designed for each mutation site in combination with the nucleotide sequences near the mutation site of the new coronavirus Indian variant, in which the 3' ends of the upstream primers match the mutation and non-mutation sites respectively. Variation point, i.e. mutated upstream primer and non-variant upstream primer, two pairs of upstream primers share one downstream primer;

[0048] When the variant site to be identified is A27S, the nucleotide sequence of the variant upstream primer is shown in SEQ ID NO.1: 5'-CCAGAACTCAATTACCCCCTT-3', and the nucleotide sequence of the non-variant upstream primer is show...

Embodiment 2

[0064] Sensitivity experiment is done to the method of the present invention (template dilution 10 6 -10 -1 copy / microliter), the experimental results are as follows:

[0065] like figure 1 As shown, when the variant site to be identified is A27S, the detection sensitivity is 7.25 copies / µl; such as figure 2 As shown, when the variant site to be identified is V213G, the detection sensitivity is 2.14 copies / µl; such as image 3 As shown, when the variant site to be identified is T376A, the detection sensitivity is 6.80 copies / µl; as Figure 4 As shown, when the variant site to be identified is R408S, the detection sensitivity is 4.21 copies / microliter.

Embodiment 3

[0067] The precision of the repeatability test evaluation method refers to the degree of closeness between a series of single measured values ​​obtained by repeated determination of the same sample under certain conditions, and is an indicator of the size of the random error. Inter-batch repeatability (intra-day, inter-day repeatability), operator / instrument repeatability, etc.

[0068] We investigated the repeatability (precision) of this method, which was measured by the coefficient of variation. The experimental results are shown in Table 1.

[0069] Table 1 Repeatability (precision) of this method

[0070]

[0071]

[0072]Coefficient of variation (%)=(Ct standard deviation / Ct mean)×100%.

[0073] It can be seen from Table 1 that:

[0074] The intra-assay coefficient of variation of A27S is in the range of 0.46%-2.19%, and the inter-assay coefficient of variation is in the range of 0.39%-2.06%;

[0075] The intra-assay coefficient of variation of V213G is in the r...

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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 Omicro variant BA-2 branch. 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. By reducing the matching degree between the variation primer and the non-variation virus nucleic acid and the matching degree between the non-variation primer and the variation virus nucleic acid in the reaction system, the amplification curve of the variation primer for amplifying the variation virus nucleic acid in the reaction system is earlier than the amplification curve of the non-variation 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 branches of novel coronavirus Omicron variant strain BA-2. 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. 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, so they need to be handed over to third-party sequencing companies for testing. The speed is slow, and there are many uncontrollable influencing factors; 2. The detection sensitivity is low. Gene amplification needs to be performed before gene sequencing is tested on the computer, and the amplification effect of low-abundance nucleic acid samples...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11
CPCC12Q1/701C12Q1/6858C12Q2600/156C12Q2535/137C12Q2521/107C12Q2531/113C12Q2561/101
Inventor 李建国成瑞玲牛嘉慧高泽峰夏娟张佳蕾张辉路哲陈佩蓉张雅卓吴长新
Owner SHANXI UNIV
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