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Triple RT-PCR primer composition for identification of H4 subtype, H6 subtype and H9 subtype AIV, and kit and use thereof

A primer combination, RT-PCT technology, applied in DNA/RNA fragments, microorganisms, recombinant DNA technology, etc., can solve the problems of inability to meet the requirements of rapid and timely diagnosis of epidemic diseases, inability to determine at the same time, and time-consuming, achieving low cost. , the effect of short time and simple operation

Pending Publication Date: 2017-07-11
LIAOCHENG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, at present, there are few detection methods for H4 subtype and / or H6 subtype and / or H9 subtype AIV at home and abroad, and the most widely used identification methods are virus isolation and sequencing, hemagglutination inhibition test, neuraminidase test, etc. Conventional methods, although these experiments have high accuracy, are time-consuming and expensive, and cannot meet the requirements for rapid and timely diagnosis of epidemic diseases during outbreaks; moreover, these detection methods cannot simultaneously determine whether the virus is H4, H6 or H9 subtype AIV

Method used

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  • Triple RT-PCR primer composition for identification of H4 subtype, H6 subtype and H9 subtype AIV, and kit and use thereof
  • Triple RT-PCR primer composition for identification of H4 subtype, H6 subtype and H9 subtype AIV, and kit and use thereof
  • Triple RT-PCR primer composition for identification of H4 subtype, H6 subtype and H9 subtype AIV, and kit and use thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: PCR primer pair design

[0055] According to the HA gene sequences of H4, H6 and H9 subtypes isolated by the laboratory in recent years, combined with the conserved sequences of the HA genes of H4, H6 and H9 subtypes published by Genbank, 3 pairs of specific primers were designed. For H4-F and H4-R, primer pair H6-F and H6-R, primer pair H9-F and H9-R, the nucleotide sequences are as follows:

[0056]

Embodiment 2

[0057] Embodiment 2: establishment and condition optimization of triple RT-PCR detection method

[0058] 1. Total viral RNA extraction

[0059] The H4N6, H6N1, and H9N2 subtype AIVs kept in the laboratory were selected, and total virus RNA was extracted respectively.

[0060] 1. Take 250 μl of virus liquid, add 750 μl Trizol, mix well, and let stand at room temperature for 5 minutes;

[0061] 2. Add 200 μl chloroform, mix by inversion, and let stand at room temperature for 5 minutes;

[0062] 3. Centrifuge at 12,000 rpm for 15 minutes at 4°C;

[0063] 4. Pipette the supernatant into a new centrifuge tube, add 500 μl isopropanol, invert and mix well, and let stand at -20°C for 20 minutes;

[0064] 5. Centrifuge at 12,000 rpm for 20 minutes at 4°C;

[0065] 6. Discard the supernatant and wash once with 1000 μl of 75% ethanol;

[0066] 7. Centrifuge at 12,000 rpm for 10 minutes at 4°C;

[0067] 8. Discard the supernatant, let it dry naturally, add 20 μl RNase-free water to ...

Embodiment 3

[0079] Embodiment 3: the specific detection of RT-PCR detection method

[0080] 1. Total RNA extraction

[0081] Select the H2N3, H4N6, H6N1, H8N4, H9N2, H10N3 subtype AIV, Newcastle disease virus, infectious laryngotracheitis virus, infectious bronchitis virus preserved in the laboratory, and extract the total RNA of the virus respectively. Method is with embodiment 2.

[0082] 2. Reverse transcription of RNA to cDNA

[0083] Method is with embodiment 2.

[0084] 3. Triple RT-PCR detection

[0085] 1. The composition of the template

[0086] A mixed solution 1 is composed of cDNA solutions of H4N6, H6N1 and H9N2 subtype AIV (volume ratio is 1:1:1), and a mixed solution 2 is composed of cDNA solutions of H4N6 and H6N1 subtype AIV (volume ratio is 1:1). A mixed solution composed of cDNA solutions of H4N6 and H9N2 subtype AIV (volume ratio 1:1) 3, a mixed solution composed of cDNA solutions of H6N1 and H9N2 subtype AIV (volume ratio 1:1) 4, H4N6 subtype AIV The cDNA solution...

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Abstract

The invention discloses a triple RT-PCR primer composition for rapid simultaneous identification of H4 subtype and / or H6 subtype and / or H9 subtype AIV. The triple RT-PCR primer composition comprises 3 pairs of specific amplification primers. The specific amplification primers comprise a primer pair of H4-F and H4-R, a primer pair of H6-F and H6-R and a primer pair of H9-F and H9-R. The invention also discloses a kit comprising the triple RT-PCR primer composition, a use of the triple RT-PCR primer composition in rapid simultaneous identification of H4 subtype and / or H6 subtype and / or H9 subtype AIV and a method for rapid simultaneous identification of H4 subtype and / or H6 subtype and / or H9 subtype AIV through the triple RT-PCR primer composition. The triple RT-PCR primer composition has the characteristics of simple operation, short detection time, strong specificity and high accuracy, provides an effective diagnostic detection scheme for low pathogenic AIV inspection and quarantine and disease monitoring and control and has an important meaning for prevention, control and monitoring of low pathogenic AIV.

Description

technical field [0001] The invention belongs to the technical field of animal virology diagnosis, in particular, it relates to a combination of triple RT-PCR primers for rapid and simultaneous identification of H4 subtype and / or H6 subtype and / or H9 subtype AIV, and also relates to the combination of the triple RT-PCR primers The combined kit and the method for quickly and simultaneously identifying H4 subtype and / or H6 subtype and / or H9 subtype AIV by using the triple RT-PCR primer combination. Background technique [0002] As we all know, in the process of poultry farming, AIV is extremely harmful to it. According to the difference in pathogenicity to chickens, AIV can be divided into highly pathogenic (highly pathogenic avian influenza virus, HPAIV) and low pathogenic poultry. Influenza virus (low pathogenic avian influenza virus, LPAIV). Among them, LPAIV includes H4, H6 and H9 subtypes, etc. LPAIV can occur all year round. After infecting poultry, there may be no obvio...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143
Inventor 李旭勇郭晶李玉保刘文强
Owner LIAOCHENG UNIV
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