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Primer pair, probe, kit and method for fluorescence quantitative PCR detection of FadV-4 type fowl adenoviruses

A fluorescent quantitative and poultry adenovirus technology, applied in the field of bioengineering, can solve the problems of SPF chicken embryo virus expansion cycle is long, the virus is difficult to detect, complex operation, etc., to achieve high sensitivity, strong specificity, good repeatability

Inactive Publication Date: 2017-06-09
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of FAdV-4 type avian adenovirus is mainly based on the traditional methods of SPF chicken embryo amplification and common PCR. However, the SPF chicken embryo amplification cycle is long, and the operation is complicated and cumbersome. It is difficult to detect, so it is urgent to establish a simple, fast and high-sensitivity detection method

Method used

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  • Primer pair, probe, kit and method for fluorescence quantitative PCR detection of FadV-4 type fowl adenoviruses
  • Primer pair, probe, kit and method for fluorescence quantitative PCR detection of FadV-4 type fowl adenoviruses
  • Primer pair, probe, kit and method for fluorescence quantitative PCR detection of FadV-4 type fowl adenoviruses

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Establishment of FAdV-4 type avian adenovirus TaqMan real-time fluorescent quantitative PCR detection method

[0027] 1. Materials

[0028] 1.1 Main reagents and instruments

[0029] Fluorescence quantitative TaqMan Universal PCR Master Mi× was purchased from Applied Biosystems; Applied Biosystems 7500 Fluorescence Quantitative Instrument; RNA reverse transcription kit Prime Script RT kit was purchased from Takara; TIANamp viral RNA extraction kit was purchased from QIAGEN.

[0030] 1.2 Virus strains

[0031] HN strain (FAdV) and DAdV-8b were clinically isolated in June 2016, and MD-CIV988, CLV-J, NDV, IBV, MG, H9N2, and H5N6 were provided by Shanghai Veterinary Research Institute.

[0032] 2. Method

[0033] 2.1 Standard plasmid construction and primer design

[0034] Refer to the 852-1518bp of the FAdV-4 avian adenovirus ON1 strain hexon gene (accession number: GU188428) in GenBank to design specific primers, FAdV-4-F1: 5'-CAACTACATCGGGTTCAGGGATAACTTC-3'...

Embodiment 2

[0048] Embodiment 2 Sensitivity and repeatability detection

[0049] Using the pGEM-Teasy-hexon plasmid as a template, press 2.28×10 7 ~2.28×10 1 Copies / μL was diluted 10 times, and the fluorescent quantitative PCR amplification was carried out, and the Ct value greater than 35 was regarded as negative. Draw a standard curve for different concentrations. The result shows: this method has very high sensitivity, the minimum detectable template concentration is 22.8copies / μL ( image 3 ), which is 100 times more sensitive than ordinary PCR. image 3 Among them, 1~7 corresponds to the concentration of standard plasmid diluted 2.28×10 by 10 times 7 ~2.28×10 1 copies / μL.

[0050] Take pGEM-Teasy-hexon plasmid 2.28×10 4 and 2.28×10 5 Copies / μL DNA of two dilutions is used as a template, and the two dilutions are repeated three times at different time periods, and each time the same template is repeated three times at the same time and three replicate holes are set, according ...

Embodiment 3

[0053] The specificity experiment of embodiment 3FAdV-4 type avian adenovirus real-time fluorescence quantitative PCR method

[0054] Collect the common virus MD-CVI988, CLV-J, MG, NDV, IBV, H9N2, H5N6, FAdV-8b virus that causes poultry disease in the laboratory, for detecting the FAdV-4 type poultry adenovirus real-time fluorescent quantitative PCR method of the present invention The specificity, system and amplification reaction conditions were amplified with reference to Example 1 above. The result is as Figure 4 As shown, MD-CVI988, CLV-J, MG, NDV, IBV, H9N2, H5N6, FAdV-8b, and sterile double distilled water have no amplification curves and are negative, and only FAdV-4 has a positive amplification curve. It shows that other viruses detected by this fluorescent quantitative PCR method have no cross-reaction with FAdV-4, further illustrating that this method has high specificity.

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Abstract

The invention discloses a primer pair, a probe, a kit and a method for fluorescence quantitative PCR detection of FadV-4 type fowl adenoviruses. The detection method takes sample DNA as a template, SEQ ID No:1 and SEQ ID No:2 as primers and SEQ ID No:3 as the probe to perform fluorescence quantitative PCR amplification and acquire a fluorescence signal. The fluorescence quantitative PCR detection method disclosed by the invention can realize quick, accurate, convenient, rapid and specific detection of FadV-4 type fowl adenoviruse nucleic acids, is high in sensitivity, strong in specificity and good in repeatability, and has wide application prospects.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a primer pair, a probe, a kit and a method for detecting FAdV-4 avian adenovirus by fluorescent quantitative PCR. Background technique [0002] Avian adenovirus (Fowl adenovirus, FAdV) belongs to Adenoviridae, according to antigenicity can be divided into A ~ E groups. Group I FAdV isolated from chickens has 12 serotypes (FAdV 1-12) and is one of the common infectious disease pathogens in chickens, ducks, geese and other birds. Since 2013, a highly pathogenic FAdV infection that can cause IBH and HPS has broken out in China, mainly affecting 3-5 week-old chicks, especially 4-week-old chicks. As of June 2015, the disease has been widespread across the country, mostly in acute onset, and mostly occurs in 20-30-day-old broiler chickens. The peak of death is 4-8 days after the onset, the disease duration is 8-15 days, and the mortality rate is 20%- 100%, causing ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 魏晓锋蔡骁垚熊炜胡建华张泉其他发明人请求不公开姓名
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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