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Polymerase chain reaction (PCR) primer pair for identifying H9 subtype avian influenza virus and application thereof

A bird flu virus, RT-PCR technology, applied in the determination/test of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. Make timely diagnosis of viruses and other problems to achieve good application prospects and strong specificity

Inactive Publication Date: 2015-02-25
LIAOCHENG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional hemagglutination inhibition test (HI) requires multiple subtypes of serum and antigens for the identification of H9 subtype AIV, which is time-consuming and laborious, and cannot make a timely diagnosis or identify the virus
There are currently no practical molecular methods for detection of H9 subtype influenza viruses

Method used

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  • Polymerase chain reaction (PCR) primer pair for identifying H9 subtype avian influenza virus and application thereof
  • Polymerase chain reaction (PCR) primer pair for identifying H9 subtype avian influenza virus and application thereof
  • Polymerase chain reaction (PCR) primer pair for identifying H9 subtype avian influenza virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0022] According to the sequence of the HA gene of the H9N2 subtype avian influenza virus, the DNAman software was used to compare the conserved region of the HA gene, and specific primers were designed for the sequence of the conserved region of the HA gene, and BLAST detection and comparison analysis was performed on Genbank , use Oligo4.0 software to analyze whether the upstream and downstream primers match, and send the qualified primers to the Beijing Synthesis Department of Shanghai Sangon Biotechnology Co., Ltd. for synthesis. 5 pairs of primers targeting HA gene were synthesized, screened with the H9N2 subtype AIV isolated and preserved in our laboratory, and the primer pairs were determined according to the amplification efficiency. Determine the upstream primer H9-F: 5'-TGTGGCAACTGAAGAAAT-3' (SEQ ID NO: 1); the downstream primer H9-R: 5'-ACCAACCTCCCTCTATGA-3' (SEQ ID NO: 2); the annealing temperature i...

Embodiment 2

[0023] The establishment of embodiment 2, RT-PCR method

[0024] 1. Extraction of total RNA

[0025] 1) Take 300 μL of chicken embryo allantoic fluid infected with H9N2 subtype avian influenza virus A / Chicken / Shandong / ZB / 2007 in a RNase-free 1.5 mL centrifuge tube, 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 700 μL) 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 the superna...

Embodiment 3

[0045] Embodiment 3, the specificity test of PCR primer pair

[0046] 1. Extraction of total RNA

[0047] 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 chicken infectious bursal disease virus, one strain of Total RNA was extracted from chicken embryo allantoic fluid of a strain of chicken Newcastle disease virus, a strain of chicken infectious bronchitis virus, and a strain of chicken infectious laryngotracheitis virus, and the method was the same as in Example 2.

[0048] 2. Reverse transcription to synthesize cDNA

[0049] The total RNA samples obtained in Step 1 were respectively reverse-transcribed using a reverse transcription kit (K1622, Fermentas) to obtain cDNA.

[0050] Reaction system, reaction conditions and method are with embodiment 2.

[0051] 3. The specificity of PCR amplification detection RT...

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Abstract

The invention discloses a polymerase chain reaction (PCR) primer pair for identifying an H9 subtype avian influenza virus (AIV) and application thereof. The PCR primer pair disclosed by the invention is composed of two single chain deoxyribonucleic acids (DNAs), wherein the two single chain DNAs are single chain DNAs shown in SEQ ID NO:1 and SEQ ID NO:2 in a sequence table. The HA gene of the H9 subtype AIV in a sample can be subjected to specific amplification, and the length of a target segment is 425bp. The method is free of cross reaction on H3, H4, H5 and other subtype AIVs, and a newcastle disease virus, avian infectious bronchitis, an infectious bursal disease virus, an infectious laryngotracheitis virus and the like; the minimum detectable quantity of virus allantoic fluid is 1*10<4.25>EID50 / 100mu L. Compared with the conventional methods such as a hemagglutination inhibition test of the virus and the like, the accordance rate of the identification result is 100%. A rapid, specific and sensitive detection means is provided for identification of the H9 subtype AIV. The PCR primer pair can be applied to rapid diagnosis of a disease caused by the H9 subtype AIV, and has a good application prospect in the aspects of clinical diagnosis and epidemiological investigation.

Description

technical field [0001] The invention relates to a pair of PCR primers for identifying H9 subtype avian influenza virus and its application. Background technique [0002] H9 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, H9 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, H9 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. In addition to infecting poultry, H9 subtype AIV can also infect humans. Therefore, establishing a detection method for H9 subtype avian influenza virus can not only quickly and accurately identify H9 subtype avian influenza virus, but also be used for epidemiol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/70C12Q2531/113
Inventor 司振书刘金华蒲娟包静楠
Owner LIAOCHENG UNIV
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