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Primer composition assisting identification of H9 subtype avian influenza virus and chicken infectious bronchitis virus and applications thereof

A technology for bronchitis and chicken infectivity, which is applied in the primer composition and application field of auxiliary identification of H9 subtype avian influenza virus and chicken infectious bronchitis virus. Carry out differential diagnosis, detect different objects and other problems, to achieve the effect of easy operation and application at the grassroots level, low cost and short time required

Inactive Publication Date: 2014-12-24
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chicken infectious bronchitis virus detection methods usually use chicken embryos for virus isolation and culture or tracheal ring tissue culture, which is a traditional detection method. It takes 10 days to use chicken embryos for virus isolation and culture, and the tracheal ring tissue culture technology is relatively difficult , not easy to operate
In short, the traditional detection method has the disadvantages of time-consuming and laborious, and it is difficult to make a quick and timely diagnosis; moreover, it cannot detect different objects in the same detection reaction, that is, it cannot perform differential diagnosis at the same time

Method used

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  • Primer composition assisting identification of H9 subtype avian influenza virus and chicken infectious bronchitis virus and applications thereof
  • Primer composition assisting identification of H9 subtype avian influenza virus and chicken infectious bronchitis virus and applications thereof
  • Primer composition assisting identification of H9 subtype avian influenza virus and chicken infectious bronchitis virus and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, the design of specific primer pair

[0033] Design a pair of primers for identifying the HA gene of the H9 subtype avian influenza virus as follows (name it as primer pair A):

[0034] H9-HA99F (Sequence 1 of the Sequence Listing): 5'-CATCGGCTACCAATCAACAAAC-3';

[0035] H9-HA571R (SEQ ID NO: 2 of the SEQUENCE LISTING): 5'-GATTATTTGTGTATTGGGCGTC-3'.

[0036] Design a pair of primers for identifying the N gene of chicken infectious bronchitis virus as follows (name it as primer pair B):

[0037] IB621F (Sequence 3 of the Sequence Listing): 5'-CAGGTAAAGGCGGAAGAAAAC-3;

[0038] IB1275R (SEQ ID NO: 4 of the SEQUENCE LISTING): 5'-TGAAGCCCATCTGGTTGAAG-3'.

Embodiment 2

[0039] Embodiment 2, the specific detection of primer pair

[0040] 1. Specificity detection of primer pair B

[0041] The experimental samples are: H4N6 subtype avian influenza virus, H3N8 subtype avian influenza virus, infectious laryngotracheitis virus, egg drop syndrome virus, Newcastle disease virus, chicken infectious bronchitis virus H120 strain and H9N2 subtype avian influenza Virus.

[0042] 1. Extract each sample to be tested (that is, the virus concentration is 10 6 EID 50 / 100 μL of virus fluid) and reverse transcribed into cDNA.

[0043] 2. Using the cDNA obtained in step 1 as a template, use primer pair B to perform PCR amplification to obtain a PCR amplification product.

[0044] PCR amplification reaction system (25 μl): 2×Mix Buffer 12.5 μl, IB621F 0.5 μl, IB1275R 0.5 μl, cDNA 2 μl, add double distilled water to 25 μl.

[0045] The reaction program of PCR amplification: 94°C for 5min; 94°C for 45s, 59°C for 45s, 72°C for 2min, 30 cycles; 72°C for 10min; 4...

Embodiment 3

[0054] Embodiment 3, the sensitivity detection of primer pair

[0055] 1. Sensitivity of primers to B

[0056] 1. Extract virus concentration to 10 5 EID 50 / 100μL, 10 4 EID 50 / 100μL, 10 3 EID 50 / 100μL, 10 2 EID 50 / 100μL or 10EID 50 / 100 μL of the total RNA of the virus liquid of chicken infectious bronchitis virus H120 strain (named as virus liquid A1, virus liquid B1, virus liquid C1, virus liquid D1 and virus liquid E1 according to the virus concentration from high to low) and reversed. Transcribed into cDNA.

[0057] 2. Using the cDNA obtained in step 1 as a template, use primer pair B to perform PCR amplification to obtain a PCR amplification product.

[0058] The reaction system of PCR amplification is the same as step 1 and 2 of embodiment 2.

[0059] The reaction procedure of PCR amplification is the same as Step 1 and 2 of Example 2.

[0060] 3. Perform 1% agarose gel electrophoresis on the PCR amplification product of step 2, see the electrophoresis ...

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Abstract

The invention discloses a primer composition assisting identification of an H9 subtype avian influenza virus and a chicken infectious bronchitis virus and applications thereof. A specific primer pair comprises a single-stranded DNA molecule shown as a sequence 3 in a sequence table and a single-stranded DNA molecule shown as a sequence 4 in the sequence table. The invention also discloses the primer composition. The primer composition comprises a primer pair A and the specific primer pair. The primer pair A comprises a single-stranded DNA molecule shown as a sequence 1 in the sequence table and a single-stranded DNA molecule shown as a sequence 2 in the sequence table. The specific primer pair can identify the chicken infectious bronchitis virus. By combining double RT-PCR, the primer composition can simultaneously identify the H9 subtype avian influenza virus and the chicken infectious bronchitis virus and has advantages of high sensitivity, high specificity, high universality, short needed time, low cost, and the like. A plurality of clinical samples can be detected. The primer composition has characteristics of simple operation, liable popularization, and convenient basic level operation and application.

Description

technical field [0001] The invention relates to a primer composition for assisting identification of H9 subtype avian influenza virus and chicken infectious bronchitis virus and application thereof. Background technique [0002] H9 subtype avian influenza and chicken infectious bronchitis are the main diseases that endanger the poultry industry in my country. H9 subtype avian influenza virus can infect a variety of poultry and mammals, and can cause disease and death in poultry, seriously affecting the healthy development of the poultry industry. Chicken infectious bronchitis is also an important infectious disease that endangers the poultry industry in my country. The sick chickens mainly show respiratory symptoms. Clinically, the symptoms of the two diseases are similar, and both virus infections lead to slow growth of broiler chickens and a decrease in egg production rate of laying hens, causing serious economic losses to the poultry industry. In addition, the variation...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/70C12Q1/68
Inventor 蒲娟刘金华魏延迪俞晨芳裴星瑶齐鲁张谞霄
Owner CHINA AGRI UNIV
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