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Method for detecting multiple-PCR (Polymerase Chain Reaction) product of influenza A virus H7N9 through mass spectrometry, and product thereof

An influenza A virus and influenza virus technology are applied in the field of primer sets and kits for detecting influenza A, and can solve the problems of no reporting scheme, difficulty in teaching detection of influenza virus, and no reporting of specific targets of influenza virus.

Active Publication Date: 2018-05-15
BIOYONG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method only briefly summarizes various possible technologies, and it neither reports specific protocols nor specific targets of influenza virus, so it is difficult to teach researchers to detect influenza virus by MALDI-TOF mass spectrometry

Method used

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  • Method for detecting multiple-PCR (Polymerase Chain Reaction) product of influenza A virus H7N9 through mass spectrometry, and product thereof
  • Method for detecting multiple-PCR (Polymerase Chain Reaction) product of influenza A virus H7N9 through mass spectrometry, and product thereof
  • Method for detecting multiple-PCR (Polymerase Chain Reaction) product of influenza A virus H7N9 through mass spectrometry, and product thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1. Primer design

[0089] In order to detect influenza A viruses, the following 9 nucleic acid sequences of influenza A viruses of 4 common subtypes, H1N1, H3N2, H5N1 and H7N9, were collected. Extract viral RNA, design reverse transcription primers for the target detection segments (M, H1, H3, H5, H7, N1, N2, N9, etc.), and reverse transcribe these fragments into cDNA, then ligate it to the plasmid vector PmdTM19- T SimpleVector was transformed, and 9 plasmids containing the 9 nucleic acid sequences were synthesized.

[0090] After identification, the plasmid DNA was extracted, and the NanoDrop ND-2000 nucleic acid detector was used to measure the concentration of the plasmid DNA, and the DNA copy number was determined as the sensitive standard product quantitative mother liquor.

[0091] In the following sequence, the sequence corresponding to the primer selected in the present invention is underlined.

[0092] (1) H1 fragment of H1N1

[0093] Influenza A virus(A / canin...

Embodiment 2

[0142] Example 2. Two-fold PCR amplification of H1N1 subtype influenza A virus

[0143] 1. Synthesize the plasmids of the H1 and M fragments of the H1N1 subtype influenza A virus, and design the specific primers corresponding to their conservative sequences. The plasmids and specific primers are all synthesized by Shanghai Jierui Bioengineering Co., Ltd. The plasmid was prepared into a 10ng / μL working solution, and the primers were prepared into a 10μM working solution. As shown in Table 1, the following primer pairs were used.

[0144] The first primer pair H1:

[0145] Upstream primer:

[0146] SEQ ID NO. 1: 5'-TGCTGGATCTGGTATTATC-3',

[0147] Downstream primer:

[0148] SEQ ID NO. 2: 5'-TGGGAGGCTGGTGTTTATAG-3';

[0149] The second primer pair M:

[0150] Upstream primer:

[0151] SEQ ID NO. 3: 5'-GGCGTTTTGAACAAACCGTC-3',

[0152] Downstream primer:

[0153] SEQ ID NO. 4: 5'-CAATCCTGTCACCTCTGACT-3'.

[0154] 2. PCR amplification

[0155] 1) The composition of the PCR reaction system is as f...

Embodiment 3

[0165] Example 3. Triple PCR amplification of H3N2

[0166] 1. Synthesize the plasmids of the H3, N2 and M three fragments of the H3N2 subtype influenza A virus, and design the specific primers corresponding to their conservative sequences. The plasmids and specific primers are all synthesized by Shanghai Jierui Biological Engineering Co., Ltd. The plasmid was prepared into a 10ng / μL working solution, and the primers were prepared into a 10μM working solution. As shown in Table 1, the following primer pairs were used.

[0167] The first primer pair H3-1:

[0168] Upstream primer:

[0169] SEQ ID NO.5: 5'-CCGGATGAGGCAACTAGTGA-3',

[0170] Downstream primer:

[0171] SEQ ID NO.6: 5'-GCAGCAAAGCCTACAGCAAC-3';

[0172] The second primer pair N2:

[0173] Upstream primer:

[0174] SEQ ID NO.9: 5'-TATCATCCCCAGTGACACAG-3',

[0175] Downstream primer:

[0176] SEQ ID NO.10: 5'-TGGGAACCAAACAAGTGTGC-3';

[0177] The third primer pair M:

[0178] Upstream primer:

[0179] SEQ ID NO. 3: 5'-GGCGTTTTGAACAAAC...

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Abstract

The invention provides a method for directly detecting a multiple-PCR (Polymerase Chain Reaction) product of an influenza A virus H7N9 through mass spectrometry. The method comprises the steps of using a specific primer for carrying out multiple-PCR amplification on virus DNA (Deoxyribonucleic Acid), and detecting the size of a fragment of the multiple-PCR product through MALDI-TOF MS (Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry). The method can be used for detecting the H7N9 hypotype influenza A virus, and can also protect a relevant detection product. The method provided by the invention is used for clinical pathogenic microorganism identification by combining multiple-PCR and the MALDI-TOF MS, and is not only fast, simple and convenient, but also easy to observe, intuitive and high in accuracy.

Description

Technical field [0001] The invention belongs to the technical field of molecular biology detection, and relates to a method and a product for detecting multiple PCR products by using characteristic peak maps of mass spectrometry. The invention also relates to a primer set and a kit for detecting influenza A by using multiple PCR amplification technology. Background technique [0002] Influenza is abbreviated as "flu", which is an acute respiratory infectious disease caused by influenza virus. It has the characteristics of sudden outbreak, rapid spread, and wide spread. It is mainly spread through coughing and sneezing. Influenza has existed for a long time in human history, as early as 1580 there was a global flu record. There were four outbreaks in the 20th century: the Spanish flu (H1N1) from 1918 to 1920, the Asian flu (H2N2) in 1957, the Hong Kong flu (H3N2) in my country in 1968 and the Russian flu in 1977 (H1N1 broke out again). In the past half century in my country (fro...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686G01N27/64
CPCG01N27/64C12Q1/686C12Q1/701C12Q2537/143
Inventor 马庆伟钟逾安娜高佳敏刘昕超王佳
Owner BIOYONG TECH
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