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H5, H7, H9 subtype auian flu virus real-time fluo rescent quantixative PCR detecting method

A real-time fluorescence quantitative, avian influenza virus technology, applied in the field of bioengineering, can solve the problems affecting the accuracy and sensitivity of test results, false positive test results, low sensitivity, etc., and achieve easy automation, good linearity, and high sensitivity. Effect

Inactive Publication Date: 2008-04-02
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its disadvantages are: since the obtained sample cDNA is extended under the action of Taq enzyme, the non-specific amplification of primers will cause false positives in the test results; and the PCR products need to be detected by electrophoresis, which is time-consuming and has low sensitivity And it is easy to cause pollution, affecting the accuracy and sensitivity of the test results

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Construction of Standard Molecules

[0030] 1. Experimental reagents

[0031] Restriction enzymes Sac I, BamH I, Pst I, EcoR I, Hind III, Kpn I, and restriction enzyme digestion corresponding buffer (Buffer) (including 10×M Buffer, 10×H Buffer, 10×L Buffer, 10 ×K Buffer) (Shanghai Haojia Technology Development Co., Ltd.);

[0032] PMD18-T vector, T 4 DNA ligase, T 4 DNA ligation buffer was purchased from Shanghai Haojia Technology Development Co., Ltd.;

[0033] dNTPs, Taq DNA polymerase and its buffer, and DL2000 Marker were purchased from Dalian Bao Biological Engineering Co., Ltd.;

[0034] Primers were synthesized by Shanghai Boya Bioengineering Co., Ltd.;

[0035] Other biochemical reagents are imported subpackages or domestic analytical pure.

[0036] 2. Experimental equipment

[0037]PTC-100 type PCR amplification instrument (MJ Research Inc.);

[0038] DNA electrophoresis analysis system includes dark box, digital camera, computer, scanner, inkjet prin...

Embodiment 2

[0067] Extraction of sample RNA

[0068] Extract the RNA of avian influenza virus in the sample according to the Trizol kit (Note: 8-10 μg of glycogen should be added during RNA precipitation, which can co-precipitate with RNA, and be dissolved in DEPC-treated water.)

[0069] 1. Experimental reagents

[0070] 1. TRIZOL reagent;

[0071] 2. DEPC treated water, RNase-free centrifuge tube and Tips:

[0072] 3. New chloroform, isopropanol and ethanol, 75% ethanol.

[0073] 2. Experimental equipment

[0074] Desktop centrifuge

[0075] 3. Experimental steps

[0076] 1). After adding 1ml Trizol solution to 200μl of the tested solution, shake vigorously, and place at room temperature for 5-10 minutes; 2). Add 200μl chloroform / isoamyl alcohol (24:1) or chloroform, shake vigorously for 30 seconds, and place at room temperature place

[0077] 3-5 minutes;

[0078] 3). Centrifuge at room temperature for 5 minutes at 12,000 rpm in a desktop centrifuge.

[0079] 4). Carefully tra...

Embodiment 3

[0085] Reverse transcription of sample RNA (RevertAid TM First Strand cDNA Synthesis Kit #K1622)

[0086] 1. Experimental reagents

[0087] 1. Reverse Transcriptase (200u / μl)

[0088] Storage buffer: 5mM Tris-HCl (pH 8.3), 0.1M NaCl, 1mM EDTA, 5mM DTT, 0.1% Triton X-100, 50% glycerol;

[0089] 2. Ribonuclease Inhibitor (20u / μl)

[0090] Storage buffer: 20mM HEPES-NaOH (pH 7.5), 50mM NaCl, 8mM DTT, 0.5mMELUGENT Detergent, 50% glycerol;

[0091] 3.5 times the volume of reaction buffer (5x Reaction Buffer)

[0092] 250mM Tris-HCl (pH 8.3, 25), 250mM KCl, 20mM MgCl 2 , 50mM DTT;

[0093] 4. dNTP Mixture (10mM dNTP Mix)

[0094] 5. Oligonucleotide primer (Oligo(dT)18Primer)

[0095] 6. DEPC-treated water (DEPC-treated Water)

[0096] 2. Experimental equipment

[0097] PTC-100 PCR Amplifier (MJ Research Inc.)

[0098] Constant temperature water bath, etc.

[0099] 3. Experimental steps

[0100] 1: RNA 1μl

[0101] Oligo(dT) 18 primer (0.5μg / μl) 1μl

[0102] +DEPC-tre...

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Abstract

This invention relates to a real time fluorescence quantitative PCR test method for H5, H7 and H9 sub-avian flu viruses including the following steps: set-up of standard molecules, the design of specific virus quantitative PCR primer and the probes, pick-up of virus sample RNA, the inverse transcription polyenzyme chain reaction to the pic-up sample RNA to get a sample cDNA, the optimization and set-up of the PCR quantitative test method for M genes.

Description

technical field [0001] The invention relates to a detection method in the technical field of bioengineering, in particular to a real-time fluorescence quantitative PCR detection method for H5, H7 and H9 subtype avian influenza viruses. Background technique [0002] Highly Pathogenic Avian Influenza (HPAI), formerly known as fowl plague, is caused by a specific subtype of influenza A virus in the Orthomyxoviridae family, usually H5 and H7 subtypes, followed by H9 subtypes Some highly pathogenic strains; its epidemiological characteristics are acute onset, rapid spread, high lethality, and can cause pathogenic blows to the breeding industry and human health. The World Organization for Animal Health (OIE) lists it as a class A infectious disease, and my country defines it as a class I animal infectious disease. There are three subtypes of avian influenza viruses, H5N1, H7N7, and H9N2, that cause human infection. Among them, H5N1 has attracted global attention due to its strong...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 张大兵杨立桃熊静静潘爱虎尹长松
Owner SHANGHAI JIAOTONG UNIV
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