RT-PCR kit for detection of poultry source pedigree H3N2 subtype canine influenza virus and application thereof

A canine influenza virus, H3N2 technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, microbial-based methods, etc., can solve the problems of inability to make rapid diagnosis, virus antigenic variation, and inability to detect viruses, etc., to achieve good results The effects of coverage and versatility, prevention of influenza pandemic, and simple operation

Active Publication Date: 2013-11-27
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Traditional canine influenza virus detection methods require virus isolation and culture and hemagglutination inhibition tests. This method is time-consuming and laborious, and ca...

Method used

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  • RT-PCR kit for detection of poultry source pedigree H3N2 subtype canine influenza virus and application thereof
  • RT-PCR kit for detection of poultry source pedigree H3N2 subtype canine influenza virus and application thereof
  • RT-PCR kit for detection of poultry source pedigree H3N2 subtype canine influenza virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, the design of specific primer pair

[0048] Using primer premier5.0, primers were designed for the highly conserved region gene of the H3N2 subtype canine influenza virus HA gene of the avian lineage.

[0049] Primers are as follows:

[0050] Upstream primer H3N2-HA-601F: 5'-CCAAGCACTAATCAAGAACAAACC-3' (SEQ ID No.1)

[0051] Downstream primer H3N2-HA-1089R: 5'-TACCATCCCTTCCCATCCATTTTCT-3' (SEQ ID No.2)

Embodiment 2

[0052] Example 2. Detection of the specificity of H3N2 subtype canine influenza virus of avian origin

[0053] The tested samples were H3N2 canine influenza virus of avian origin (A / canine / Beijing / 364 / 2009), seasonal H3N2 human influenza virus (A / Jiangxi / 262 / 2005), H3N8 canine influenza virus of horse origin (A / canine / California / 70645-4 / 2006), H1N1 canine influenza virus (A / canine / Beijing / cau9 / 2009), processed negative swab samples and double distilled water negative control solution.

[0054] 1. Extraction of total RNA by Trizol method

[0055] (1) Take 300 μl of each sample, add 900 μl Trizol to each tube, and gently invert and mix 10 times.

[0056] (2) Add 200 μl of chloroform, invert and mix 10 times, and ice-bath for 5-10 minutes, during which time gently invert and mix. Centrifuge at 13000rpm for 15min at 4°C.

[0057] (3) Transfer 700 μl of the supernatant into a new centrifuge tube, add an equal amount of isopropanol, and gently invert to mix. After mixing, place ...

Embodiment 3

[0078] Example 3. Detection of sensitivity of avian-derived lineage H3N2 subtype canine influenza virus

[0079] The detection samples of H3N2 subtype canine influenza virus of avian origin were: A / canine / Beijing / 364 / 2009(H3N2), A / canine / Beijing / 253 / 2009(H3N2), A / canine / Beijing / 305 / 2009( H3N2), A / canine / Beijing / 420 / 2010(H3N2), A / canine / Beijing / 511 / 2010(H3N2), A / canine / Liaoning / 1578 / 2010(H3N2), A / canine / Liaoning / 1585 / 2010 (H3N2), A / canine / Beijing / 1215 / 2012 (H3N2), A / canine / Beijing / 0108 / 2013 (H3N2).

[0080] 1. Extraction of total RNA by Trizol method

[0081] Extract the total RNA of the test sample, and the method is the same as step 1 in Example 2.

[0082] 2. Synthesis of cDNA by reverse transcription

[0083] The method is the same as step 2 in Example 2.

[0084] 3. PCR amplification

[0085] The method is the same as Step 3 in Example 2.

[0086] 4. Agarose gel electrophoresis

[0087] The method is the same as Step 4 in Example 2.

[0088]Electrophoresis results...

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Abstract

The invention discloses an RT-PCR kit for detection of poultry source pedigree H3N2 subtype canine influenza virus and application thereof. The invention provides a primer pair for assisting detection of poultry source pedigree H3N2 subtype canine influenza virus. The nucleotide sequence of one primer is shown as a SEQ ID No.1, and the nucleotide sequence of the other primer is shown as a SEQ ID No. 2. Detection of poultry source pedigree H3N2 subtype canine influenza virus by the kit provided by the present invention is faster, and has higher singularity, sensibility and sensitivity than a traditional method. The kit can detect virus allantoic fluid as well as clinically acquired samples, and is easy for popularization as a useful tool for diagnosis and epidemiological investigation of poultry source pedigree H3N2 subtype canine influenza virus.

Description

technical field [0001] The invention relates to a RT-PCR reagent kit for detecting H3N2 subtype canine influenza virus of avian origin and application thereof. Background technique [0002] Canine influenza is a highly contagious disease of dogs caused by canine influenza virus. Affected dogs often show cold symptoms such as cough and pneumonia. In recent years, the reports of canine influenza virus infection have increased rapidly. Dogs are important companion animals for human beings. People pay more and more attention to the health of dogs. In addition, influenza viruses in dogs may increase the chance of infecting humans through close human-dog contact. Therefore, canine influenza virus not only affects the health of dogs, but also poses a serious threat to public health security. [0003] It has been reported in the literature that dogs can be infected with avian-derived H3N2 subtype canine influenza virus, equine-derived H3N8 subtype canine influenza virus, H1N1 inf...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 孙怡朋刘金华王倩
Owner CHINA AGRI UNIV
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