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A dual-labeled probe real-time fluorescent PCR method for rapid identification and detection of serotype 4 avian adenovirus

A technology for identification and detection of avian adenovirus, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of polluted environment, inaccurate quantification, complicated operation, etc. The effect of avoiding false negative/positive situations

Active Publication Date: 2020-11-24
山东农科种业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The entire experimental process of this technology is carried out in a completely closed tube state, without the need for PCR post-processing and tedious electrophoresis, ultraviolet or staining detection, which solves the problems of inaccurate quantification, complicated operation, and environmental pollution in conventional PCR experiments.

Method used

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  • A dual-labeled probe real-time fluorescent PCR method for rapid identification and detection of serotype 4 avian adenovirus
  • A dual-labeled probe real-time fluorescent PCR method for rapid identification and detection of serotype 4 avian adenovirus

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Embodiment 1

[0024] 1. Primer Design

[0025] Referring to the genome sequence of FAV in GenBank, a pair of primers (FAV-FQ-F, FAV-FQ-R) and probe P were designed, and the fluorescent markers were FAM and Eclipse; the primers were synthesized by Dalian TaKaRa Company and purified at HPLC level; The primer and probe sequences are as follows:

[0026] FAV-FQ-F: 5'-CTAGGGTTCTGAACTTTG-3' (48-65),

[0027] FAV-FQ-R: 5'-CGGTAAACATTTCAAGGA-3' (190-173), the amplified length is 143bp;

[0028] Probe P: 5'-(FAM)TGACGCCAGTTTCGCTTTCG (Eclipse)-3'(72-91).

[0029] 2. Real-time PCR

[0030] 2.1 Extraction of DNA

[0031] Broiler FAV isolated strains GF16 and WF816 were selected for detection. Virus DNA was extracted according to the operation of TaKaRa DNAiso reagent, and finally 30 μL of ultrapure water was used to dissolve the DNA; the specific operation was as follows:

[0032] Inoculate the yolk sac of 6-day-old SPF chicken embryos with the virus to be tested, collect the allantoic fluid and e...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a double-marker probe real-time fluorescent PCR (polymerase chain reaction) method for quickly identifying Type IV FAC (fowl adenovirus) in serum. The method comprises the following steps of firstly, according to a sequence of a fowl adenovirus genome, designing a pair of primers FAV-FQ-F and FAV-FQ-R and a probe P; then, extracting DNA (deoxyribonucleic acid) of the FAV virus, and dissolving by pure water; finally, using the designed primers and probe to perform real-time fluorescent PCR detection. The method has the advantages that the Type IV FAV in serum can be quickly, sensitively and accurately detected, the detection time is greatly shortened, and the other epidemic main serum types in the corresponding period can be distinguished; the false negative / positive condition of the common PCR is avoided, the rapidity, accuracy, sensitivity and good repeatability are realized, and the method can be applied to laboratory detection of FAV samples.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a detection method for avian adenovirus, in particular to a double-labeled probe real-time fluorescent PCR method for rapidly identifying and detecting serum type 4 avian adenovirus. Background technique [0002] Fowl adenovirus (FAV) can be divided into 3 groups according to antigenic differences, among which group I is traditional adenovirus, including 12 serotypes (FAV1-12) isolated from chickens, 2 serotypes from turkey serotypes, goose 3 serotypes and duck 2 serotypes. Avian adenovirus can cause inclusion body hepatitis, pericardial effusion, gizzard erosion and other lesions. Since June 2015, an infectious disease characterized by rapid death, high mortality, and pericardial effusion as the main lesion has broken out in broilers in some areas of Shandong Province. The infection speed of the disease is very fast, and the infected hosts have also expanded to breeds such as laying...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686
CPCC12Q1/686C12Q1/701C12Q2563/107C12Q2561/113
Inventor 袁小远王友令于可响艾武亓丽红
Owner 山东农科种业科技有限公司