Combined nucleic acid real-time fluorescent detection method for influenza A H1N1 virus and influenza A virus and kit

A type of influenza A virus and influenza virus technology, applied in the biological field, can solve the problem of high proportion of infectious and recessive infection, and achieve the effects of shortening operation time, reducing cost and reducing labor intensity

Inactive Publication Date: 2012-10-03
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fourth, flu patients can detoxify the day before the onset of the disease. Some people do not get sick after infection, but they are still contagious, and the proportion of recessive infection is quite high.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Design of specific primers and probes for Influenza A virus and Influenza A H1N1 virus

[0057] at NCBI http: / / www.ncbi.nlm.nih.gov / genomes / FLU / Database / request.cgi Search out the MP gene fragment sequences of all influenza A viruses, and find out the conserved segments of influenza A viruses through multiple comparisons. Primers and probes were designed on the conserved fragments using Express Primer.

[0058] Primers and probes were designed using Express Primer for the PA gene fragment sequence of influenza A H1N1 (2009 epidemic). Compare the designed primers and probes with all viral sequences to find the most variable primers and probes.

[0059] The full-length PA gene sequence of the above-mentioned type A H1N1 (popular in 2009) is derived from NCBI (>gi|227831814|gb|FJ966977.1|InfluenzaAvirus (A / California / 07 / 2009(H1N1)) segment 3 polymerase PA(PA) gene, complete cds)

[0060] The design result is:

[0061] Specific primers for influenza A virus ...

Embodiment 2

[0072] Embodiment two: establish and optimize the quantitative PCR reaction system and condition of influenza A virus and influenza A H1N1 influenza virus

[0073] Establish a 50 μl reaction system with the following component concentrations: 25 μl of 2x RT-PCR buffer, 5 U of Taq enzyme, 5 U of reverse transcriptase, 0.2 μM of upstream and downstream primers, 0.15 μM of probe, 1 μl of ROX calibration solution (TaKaRa company) , 4 μl of extracted RNA;

[0074] Establish the following reaction conditions: 42°C for 5 minutes → 95°C for 10 seconds → 95°C for 5 seconds → 60°C for 31 seconds, wherein 40 cycles were performed between 95°C for 5 seconds → 60°C for 31 seconds, and the FAM on the fluorescent PCR instrument was selected. to test.

[0075] Reverse transcription and real-time fluorescent quantitative PCR amplification

[0076] Take 2 μl or 4 μl of extracted RNA as the template for RT-PCR reaction, and add 20 μl or 50 μl of RT-PCR reaction solution to the eight-tube tube ...

Embodiment 3

[0099] Example 3: Establishment of real-time fluorescence detection method

[0100] The instrument that the present invention adopts has:

[0101] Thermal Cycler DiceTM Real Time System (TaKaRa)

[0102] Smart System (Cepheid)

[0103] ABI PRISM 7000 / 7700 / 7900HT, 7300 / 7500 Real-Time PCR System, 7500Fast Real-Time PCR System (Applied Biosystems)

[0104] Line-Gene (Bioer, Hangzhou Bioer)

[0105] LightCycler (Roche Diagnostics)

[0106] Mx3000P (Stratagere) and various other Real Time PCR amplification instruments.

[0107] Result judgment:

[0108] Take the use of ABI7300 fluorescent quantitative PCR instrument as an example, and refer to the instructions of each instrument for operations on other instruments.

[0109] After the reaction, adjust the Start value of Baseline according to the analyzed image, adjust the amplification curve of the negative control to be flat or below the threshold line, and click Analysis to automatically obtain the analysis result.

[011...

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PUM

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Abstract

The invention provides a combined nucleic acid real-time fluorescent detection method for simultaneously detecting an influenza A H1N1 virus and an influenza A virus and a kit. The combined nucleic acid real-time fluorescent detection method for the influenza A H1N1 virus and the influenza A virus comprises the following steps of: (1) extracting virus RNA; (2) carrying out fluorescent quantitative PCR (Polymerase Chain Reaction) detection; and (3) judging a detecting result. By carrying out multiple sequence comparison and aiming at a conserved gene fragment of the influenza A virus and the influenza A H1N1 virus (infecting in 2009), a primer and a probe with high specificity are designed and used for real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection. The invention can be used for detecting influenza A virus RNA of human influenza, swine influenza, avian influenza, and other influenza viruses, meanwhile specifically detecting the influenza A H1N1virus (infecting in 2009) RNA and carrying out double analysis so that a detecting result is more reliable.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a real-time fluorescent detection method and a kit for simultaneously detecting Type A and Type A H1N1 influenza viruses. Background technique [0002] Influenza, referred to as influenza, is an acute respiratory infectious disease caused by three types of influenza viruses: A, B, and C. According to its surface structure (hemagglutinin H and neuraminidase N) and its genetic characteristics, influenza A virus can be divided into many subtypes. Up to now, influenza A virus has discovered 16 subtypes of hemagglutinin (H1-H16), 9 subtypes of neuraminidase (N1-N9). Influenza A (H1N1) virus is one of these subtypes and it causes influenza A (H1N1). [0003] In 2009, the influenza A (H1N1) influenza epidemic, which broke out from Mexico and spread to many countries around the world, was caused by a new variant of the influenza A (H1N1) virus, which was called a "public health emergency of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 曾令文顿博影刘杰
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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