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Probe and primer pair assisting identification of drug resistance of influenza A virus H3N2

A H3N2, influenza virus technology, applied in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of low detection sensitivity, large consumption, cumbersome experimental operation, etc., to achieve rapid detection, increase Reliability, effectiveness in reducing false positive results

Active Publication Date: 2013-07-10
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Real-time PCR technology solves the problem that traditional PCR is difficult to quantify the initial template of amplification, avoids the shortcomings of traditional detection technology such as large sample consumption, cumbersome experimental operation, and low detection sensitivity, and has the characteristics of rapidity and avoidance of cross-contamination. Can be widely used in gene detection, gene expression, SNP analysis and other fields

Method used

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  • Probe and primer pair assisting identification of drug resistance of influenza A virus H3N2
  • Probe and primer pair assisting identification of drug resistance of influenza A virus H3N2
  • Probe and primer pair assisting identification of drug resistance of influenza A virus H3N2

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The design of embodiment 1, primer and probe

[0030] Primers and probes were designed according to the NA genes of influenza A H3N2 viruses from 2005 to 2010 published by GeneBank. The primers and probes are aimed at the 292nd amino acid mutation (R292K) of the NA protein of influenza A H3N2 virus (GENBANK ACCESSION NO.ABY25771). Among the probes, FAM is a fluorescent reporter group, is a fluorescence quenching group.

[0031] R292K-P (probe): 5'-FAM-CTGCAAAGACAAYTGG-MGB-NFQ-3' (Y is T or C; sequence 1);

[0032] R292K-F (upstream primer): 5'-TCGATATCCTGRTGTCAGATGTG-3' (R is A or G; Sequence 2);

[0033] R292K-R (downstream primer): 5'-TTATATCTACKATGGGCCTATTGGA-3' (K is G or T; SEQ ID NO: 3).

Embodiment 2

[0034] Embodiment 2, preparation and application of kit A

[0035] 1. Preparation of Kit A

[0036] Kit A consists of the following primers and probes:

[0037] R292K-F (upstream primer): 5'-TCGATATCCTGGTGTCAGATGTG-3';

[0038] R292K-R (downstream primer): 5'-TTATATCTACGATGGGCCTATTGGA-3'.

[0039] R292K-P (probe): 5'-FAM-CTGCAAAGACAACTGG-MGB-NFQ-3'.

[0040] 2. Preparation of Standards

[0041]Standard RNA A-1 and standard RNA A-2 correspond to NA protein cDNA (GENBANK ACCESSION NO. ABY25771 from nucleotides 838 to 1255 at the 5' end, and 875th is the mutation site that causes R292K). There is only one nucleotide difference between standard RNA A-1 and standard RNA A-2 at the mutation site, and standard RNA A-1 is wild type (the 38th nucleotide from the 5' end is G), The standard RNA A-2 is a drug-resistant type (the 38th nucleotide from the 5' end is A).

[0042] 1. Preparation of standard RNA A-1

[0043] (1) DNA shown in sequence 4 of the sequence listing was synthes...

Embodiment 3

[0070] Embodiment 3, preparation and application of kit B

[0071] 1. Preparation of Kit B

[0072] Kit B consists of the following primers and probes:

[0073] R292K-F (upstream primer): 5'-TCGATATCCTGGTGTCAGATGTG-3';

[0074] R292K-R (downstream primer): 5'-TTATATCTACTATGGGCCTATTGGA-3'.

[0075] R292K-P (probe): 5'-FAM-CTGCAAAGACAATTGG-MGB-NFQ-3'.

[0076] 2. Preparation of Standards

[0077] Standard RNA B-1 and Standard RNA B-2 correspond to NA protein cDNA (GENBANK ACCESSION NO. ABY25771 from nucleotides 838 to 1255 at the 5' end, and 875th is the mutation site that causes R292K). There is only one nucleotide difference between the standard RNA B-1 and the standard RNA B-2 at the mutation site, and the standard RNA B-1 is wild type (the 38th nucleotide from the 5' end is G), The standard RNA B-2 is a drug-resistant type (the 38th nucleotide from the 5' end is A).

[0078] 1. Preparation of standard RNA B-1

[0079] (1) DNA shown in sequence 8 of the sequence listin...

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Abstract

The invention discloses a probe and a primer pair assisting identification of drug resistance of influenza A virus H3N2. A nucleotide sequence of the probe is shown as a sequence 1 in the sequence table; a nucleotide sequence of the specific primer pair is comprised of a NDA shown in a sequence 2 and a DNA shown in a sequence 3; The probe and the primer pair can be used for assisting identification of a No. 292 site amino acid mutation on a NA protein of the influenza A virus H3N2, so as to assist identification of drug resistance of the influenza A virus H3N2. The invention overcomes defectsof large sample consumption, complex experiment operation and low detection sensitivity of a traditional detection technology; the application of the probe further enhanced detection singularity and result reliability; and a detection sensitivity of the present invention can reach 500copies / ul. In addition, the invention is especially suitable for science research, clinic and virus epidemiology monitoring, etc.

Description

technical field [0001] The invention relates to a probe and a pair of primers for assisting in identifying the drug resistance of influenza A (H3N2) virus. Background technique [0002] 1. Influenza A virus and human diseases [0003] Influenza viruses belong to the Orthomyxoviridae family and are divided into three types: A, B, and C. Influenza A and B pose a greater threat to humans. Among them, influenza A antigens mutate frequently, which can cause a worldwide pandemic and pose the greatest threat to humans. . The clinical manifestations of diseases caused by such viruses are acute onset, high fever, myalgia, headache accompanied by severe discomfort, dry cough, and sore throat. In severe cases, it may lead to death and seriously threaten human health. [0004] 2. Detection of drug-resistant mutations in the NA protein of influenza A (H3N2) virus [0005] Anti-influenza virus drugs are effective measures to treat and alleviate influenza symptoms, but influenza viruses...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 秦成峰姜涛刘娟韩剑峰秦鄂德
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI