Mass spectrum method for detecting H3N2 fragment multiple PCR product and its product

A mass spectrometry, multiplex technology, applied in the direction of biochemical equipment and methods, DNA / RNA fragments, microbial determination / inspection, etc., can solve the problem of no specific target of influenza virus reported, no reporting scheme, difficult to teach and detect influenza virus, etc. question

Active Publication Date: 2018-08-03
BIOYONG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Mass spectrum method for detecting H3N2 fragment multiple PCR product and its product
  • Mass spectrum method for detecting H3N2 fragment multiple PCR product and its product
  • Mass spectrum method for detecting H3N2 fragment multiple PCR product and its product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1. Primer Design

[0088] In order to detect influenza A virus, the following nine nucleic acid sequences of four common subtypes of influenza A virus, 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.), reverse transcribe these segments into cDNA, and connect them to the plasmid vector PmdTM19- Transformation was carried out on T SimpleVector, and 9 plasmids respectively containing the 9 nucleic acid sequences were synthesized.

[0089] After identification, the plasmid DNA was extracted, and the concentration of the plasmid DNA was measured using a NanoDrop ND-2000 nucleic acid detector, and the copy number of the DNA was determined as a sensitivity standard to quantify the mother liquor.

[0090] In the following sequences, the sequences corresponding to the selected primers of the present invention are underlined.

[0091] (1) H1 fragment...

Embodiment 2

[0141] 2-fold PCR amplification of embodiment 2.H1N1 subtype influenza A virus

[0142] 1. Synthesize the plasmids of the H1 and M fragments of the H1N1 subtype influenza A virus, and design specific primers corresponding to their conserved sequences. The plasmids and specific primers are all synthesized by Shanghai Jierui Bioengineering Co., Ltd. The plasmid was made into a 10ng / μL working solution, and the primer was made into a 10μM working solution. As shown in Table 1, the following primer pairs were used.

[0143] First primer pair H1:

[0144] Upstream primers:

[0145] SEQ ID NO.1: 5'-TGCTGGATCTGGTATTATC-3',

[0146] Downstream primers:

[0147] SEQ ID NO.2: 5'-TGGGAGGCTGGTGTTTATAG-3';

[0148] Second primer pair M:

[0149] Upstream primers:

[0150] SEQ ID NO.3: 5'-GGCGTTTTGAACAAACCGTC-3',

[0151] Downstream primers:

[0152] SEQ ID NO. 4: 5'-CAATCCTGTCACCTCTGACT-3'.

[0153] 2. PCR amplification

[0154] 1) The composition of the PCR reaction system is a...

Embodiment 3

[0164] Triple PCR amplification of embodiment 3.H3N2

[0165] 1. Synthesize the plasmids of the H3, N2 and M segments of the H3N2 subtype influenza A virus, and design specific primers corresponding to their conserved sequences. The plasmids and specific primers are all synthesized by Shanghai Jierui Bioengineering Co., Ltd. The plasmid was made into a 10ng / μL working solution, and the primer was made into a 10μM working solution. As shown in Table 1, the following primer pairs were used.

[0166] First primer pair H3-1:

[0167] Upstream primers:

[0168] SEQ ID NO.5: 5'-CCGGATGAGGCAACTAGTGA-3',

[0169] Downstream primers:

[0170] SEQ ID NO.6: 5'-GCAGCAAAGCCTACAGCAAC-3';

[0171] Second primer pair N2:

[0172] Upstream primers:

[0173] SEQ ID NO.9: 5'-TATCATCCCCAGTGACACAG-3',

[0174] Downstream primers:

[0175] SEQ ID NO.10: 5'-TGGGAACCAAAACAAGTGTGC-3';

[0176] Third primer pair M:

[0177] Upstream primers:

[0178] SEQ ID NO.3: 5'-GGCGTTTTGAACAAACCGTC-3',...

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PUM

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Abstract

The invention provides a method for directly detecting multiple PCR product of influenza A virus through a mass spectrum method. The method uses a specific primer for multiple PCR amplification on virus DNA, and is capable of detecting a fragment size of a multiple PCR product through MALDI-TOF. The method can be used for detecting H3N2 subtype influenza A virus. The method can protect the relative detection products. The method can be used for clinical pathogenic organism identification by combining with multiple PCR and a MALDI-TOF MS phase, and has the advantages of rapidity, simpleness, easy observation, direct viewing, and high accuracy.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, and relates to a method for detecting multiple PCR products using a characteristic peak map of mass spectrometry and a product thereof. The present invention also relates to a primer set and a kit for detecting influenza A using the multiplex PCR amplification technology. Background technique [0002] Influenza, referred to as "flu", is an acute respiratory infectious disease caused by influenza virus. Influenza has been around for a long time in human history, with worldwide records of influenza as early as 1580. There were four outbreaks in the 20th century: the Spanish flu (H1N1) in 1918-1920, the Asian flu (H2N2) in 1957, the Hong Kong flu in 1968 (H3N2) and the Russian flu in 1977 (H1N1 broke out again). In the past half century (since 1953), there have been 17 large, medium and small-scale influenza outbreaks in my country, of which 2 were pandemics. [0003] The viru...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11G01N30/72
CPCC12Q1/686C12Q1/701G01N30/72C12Q2537/143
Inventor 马庆伟钟逾安娜高佳敏刘昕超王佳
Owner BIOYONG TECH
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