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Dual reverse transcription-polymerase chain reaction (RT-PCR) detection method for identifying H9N2 subtype avian influenza virus

A bird flu virus and auxiliary identification technology, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve the problems of inability to identify viruses or make a diagnosis, time-consuming and labor-intensive, and achieve strong specificity , the effect of good application prospects

Inactive Publication Date: 2014-01-29
LIAOCHENG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional hemagglutination inhibition test (HI), neuraminidase inhibition test (NI), etc. require multiple subtypes of serum and antigens, especially NI test, which is time-consuming and laborious, and cannot identify or diagnose the virus in time
Currently there is no molecular biology method for the simultaneous detection of H9 and N2 subtypes of influenza virus

Method used

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  • Dual reverse transcription-polymerase chain reaction (RT-PCR) detection method for identifying H9N2 subtype avian influenza virus
  • Dual reverse transcription-polymerase chain reaction (RT-PCR) detection method for identifying H9N2 subtype avian influenza virus
  • Dual reverse transcription-polymerase chain reaction (RT-PCR) detection method for identifying H9N2 subtype avian influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1, design, synthesis and screening of primers

[0022] According to the sequences of the HA and NA genes of the H9N2 subtype avian influenza virus, the conserved regions of the HA and NA genes were found by comparison with the DNAman software, and specific primers were designed for the conserved regions of the HA and NA genes, and published in Genbank BLAST detection comparison analysis was carried out on the Internet, and Oligo4.0 software was used to analyze whether the upstream and downstream primers matched, and the qualified primers were sent to the Beijing Synthesis Department of Shanghai Sangong Biotechnology Co., Ltd. for synthesis. Five pairs of primers targeting HA and NA genes were synthesized, screened with the H9N2 subtype AIV isolated and preserved in our laboratory, and the combination of primer pairs was determined according to the amplification efficiency. For the upstream primer H9-F of the H9 gene: 5'-CATAATGGGATGCTGTGTGC-3' (SEQ ID NO: 1);...

Embodiment 2

[0023] Embodiment 2, establishment of double RT-PCR method

[0024] 1. Extraction of total RNA

[0025] 1) Take 300 μL of allantoic fluid from chicken embryos infected with H9N2 subtype avian influenza virus A / Chicken / Shandong / ZB / 2007 and A / Chicken / Shandong / 6 / 96 respectively, and add 900 μL Trizol reagent (Invitrogen), the ratio of the two is 1:3, gently invert and mix 10 times to completely dissolve the nucleoprotein complex;

[0026] 2) Add 200 μL of chloroform (chloroform), invert and mix 10 times, place in an ice bath for 5 minutes, during which time gently invert and mix; centrifuge at 12,000 rpm at 4°C for 15 minutes.

[0027] 3) Transfer the aqueous phase (about 700μL) to a new RNase-free 1.5mL centrifuge tube, add an equal amount of isopropanol, invert and mix several times, place at -20°C for 10 minutes, then centrifuge at 12,000rpm at 4°C for 15 minutes , discard the supernatant;

[0028] 4) Wash with 1 mL of 75% RNase-free ice ethanol, centrifuge briefly, discard...

Embodiment 3

[0044] Embodiment 3, the specificity test of double RT-PCR detection method

[0045] 1. Extraction of total RNA

[0046] One strain of H9N2 subtype avian influenza virus, one strain of H3N8 subtype avian influenza virus, one strain of H4N6 subtype avian influenza virus, one strain of H5N1 subtype avian influenza virus, one strain of H5N8 subtype avian influenza virus, and one strain of chicken Newcastle disease Total RNA was extracted from chicken embryo allantoic fluid of virus and a strain of chicken infectious bronchitis virus, and the method was the same as in Example 2.

[0047] 2. Reverse transcription to synthesize cDNA

[0048] The total RNA samples obtained in Step 1 were respectively reverse-transcribed using a reverse transcription kit (K1622, Fermentas) to obtain cDNA.

[0049] Reaction system, reaction conditions and method are with embodiment 2.

[0050] 3. Specificity of PCR amplification detection double RT-PCR method

[0051] Using the primer pair combinat...

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Abstract

The invention discloses a dual reverse transcription-polymerase chain reaction (RT-PCR) detection method for identifying an H9N2 subtype avian influenza virus. A pair of primers is designed respectively according to HA genes and NA genes of the H9N2 subtype of avian influenza virus (AIV), the HA genes and NA genes of the H9N2 subtype AIV in a sample can be subjected to specific amplification, and lengths of target fragments are respectively 700bp (HA) and 423bp (NA). According to the method, cross reaction is avoided in subtype AIV such as H3N8, H4N6 and H5N8, Newcastle disease virus and infectious bronchitis virus of chicken; the lowest detection amount of allantoic fluid of the virus is 1*103.25EID50 / 100uL; compared with conventional methods such as hemagglutination inhibition of virus and a neuraminidase inhibition test, the method has the advantage that the coincidence rate of the identification result is 100 percent. A rapid, specific and sensitive detection means is provided for identifying the H9N2 subtype AIV. The detection method can be used for rapidly diagnosing diseases caused by the H9N2 subtype AIV and has good application prospects in aspects of clinical diagnosis and epidemiological investigation.

Description

technical field [0001] The invention relates to a double RT-PCR detection method for identifying H9N2 subtype avian influenza virus. Background technique [0002] H9N2 subtype avian influenza virus is a low pathogenicity influenza virus. In 1994, H9N2 subtype AIV was isolated for the first time in Guangdong, my country. For more than ten years, H9N2 subtype avian influenza has been on the rise in my country, especially in concurrent or When secondary bacterial infection occurs, serious economic losses can be caused. At present, H9N2 subtype AIV exists widely in my country, and it is the main AIV subtype affecting the poultry industry in my country. Seriously affect the development of my country's poultry industry. Studies have shown that the H9N2 subtype AIV provided the internal gene for the H5N1 subtype AIV that caused the Hong Kong flu in 1997. In addition to infecting poultry, H9N2 subtype AIV can also infect humans. Therefore, to establish a detection method for H9N2...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/70C12Q1/686C12Q2537/143C12Q2531/113
Inventor 司振书刘金华蒲娟魏延迪包静楠
Owner LIAOCHENG UNIV
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