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

A technology of coronavirus and mutant strains, applied in the biological field, can solve the problems affecting the detection timeliness, throughput and cost of virus mutant strains, high cost of testing materials and labor, and many uncontrollable influencing factors, so as to achieve easy high-throughput operation , Reduce the probability of operational contamination, and improve the effect of detection throughput

Pending Publication Date: 2022-04-05
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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-abun

Method used

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

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Experimental program
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Effect test

Embodiment 1

[0053] A qRT-PCR method for identifying novel coronavirus Lambda mutant strains, comprising the following steps:

[0054] Step 1, use the Trizol method to extract the RNA of the new coronavirus;

[0055] Step 2, ARMS-based qRT-PCR primer probe design:

[0056] 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;

[0057] When the variable site to be identified is G75V, the nucleotide sequence of the mutated upstream primer is shown in SEQ ID NO.1: 5'-CATGTCTCTGGGACCAACGT-3', and the nucleotide sequence of the non-variant upstream primer is shown in SE...

Embodiment 2

[0079] Method of the present invention is done sensitivity experiment (template dilution 10 7 -10 0 copy / microliter), the experimental results are as follows Figure 1-7 As shown, when the variable site to be identified is G75V, the detection sensitivity is 5.63 copies / microliter; when the variable site to be identified is T76I, the detection sensitivity is 9.10 copies / microliter; when the variable site to be identified is When it is △246-252, the detection sensitivity is 3.85 copies / microliter; when the mutation site to be identified is L452Q, the detection sensitivity is 4.02 copies / microliter; when the mutation site to be identified is F490S, the detection sensitivity is 6.39 copies / microliter; when the mutation site to be identified is D614G, the detection sensitivity is 2.47 copies / microliter; when the mutation site to be identified is T859N, the detection sensitivity is 4.16 copies / microliter.

Embodiment 3

[0081] The precision of the repeatability test evaluation method refers to the closeness between a series of single measurement 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.

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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 Lambda 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 mutant strains of novel coronavirus Lambda. 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 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 detecti...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
Inventor 李建国高泽峰杨友袁悉梅吴长新
Owner SHANXI UNIV
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