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A primer pair for preparing and detecting avian adenovirus type 4 kit and its application

A detection kit and avian adenovirus technology, which are applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as the increase in reported cases and economic losses in the chicken industry, and achieve accurate results. , the effect of high detection sensitivity

Active Publication Date: 2019-04-02
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since 2012, the number of reported cases of the disease has increased nationwide, and since June 2015, it has become popular and broke out in Jiangsu, Anhui, Shandong, Liaoning, Henan, Inner Mongolia and other places on a large scale. The chicken industry has brought great economic losses

Method used

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  • A primer pair for preparing and detecting avian adenovirus type 4 kit and its application
  • A primer pair for preparing and detecting avian adenovirus type 4 kit and its application
  • A primer pair for preparing and detecting avian adenovirus type 4 kit and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 DNA Extraction

[0044] 1. Sample processing

[0045] Treatment of tissue samples: Take tissues that are easy to separate FAdV4, such as the liver and spleen of dead chickens, cut them into pieces with scissors, add sterilized PBS at a volume ratio of 1:3, and grind them thoroughly. The homogenized tissue suspension was repeatedly frozen and thawed at -20°C (or lower)-room temperature for 3 times, then centrifuged at 12,000g for 10min, and 200μL of the supernatant was taken into a new sterilized centrifuge tube.

[0046] Allantoic fluid treatment: collect the allantoic fluid from virus-infected chicken embryos and centrifuge at 12,000 g for 10 min, and take 200 μL of the supernatant into a new sterilized centrifuge tube. At the same time, the allantoic fluid of uninfected chicken embryos was collected as a negative sample; the allantoic fluid infected with FAdV4ZJ2015-1 was used as a positive control.

[0047] 2. DNA extraction

[0048] Take 200 μL of the a...

Embodiment 2

[0049] Embodiment 2 PCR reaction

[0050] 1. Primer design

[0051] According to the conserved sequence on the FAdV4 structural protein Hexon gene, the following primers were designed and synthesized:

[0052] FAdV-F is 5'-ATACCAACACGAGCACCTC-3';

[0053] FAdV-R is 5'-TTATCCCTGAACCCGATG-3';

[0054] It is expected to amplify a 403bp DNA fragment of avian adenovirus. The sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0055] 2. PCR reaction

[0056] Take 2.5 μL of extracted DNA, add 2 μL each of 10 uM primer FAdV-F and 10 uM primer FAdV-R, 2.5 mM dNTP 4 μL, 10×buffer 5 μL, template 2.5 μL, add water to 50 μL; : Pre-denaturation at 95°C for 5min, denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, 35 cycles, and extension at 72°C for 10min for PCR amplification.

[0057] The PCR products were subjected to 1.5% agarose gel electrophoresis, stained with DueRed, and observed under ultraviolet light. The result is as figur...

Embodiment 3

[0060] Example 3 Specificity verification of the kit

[0061] Take the extracted DNA and common poultry viruses from 3 positive disease samples from Shandong obtained by our laboratory, including other serotypes of adenovirus (type 1 and type 5), avian influenza virus, chicken Newcastle disease virus, chicken infectious bronchitis virus The DNA extracted from the allantoic fluid of infected chicken embryos was subjected to PCR under the same conditions.

[0062] The result is as figure 2 As shown, only the target DNA fragments of the expected size were amplified from the DNA extracted from the three disease materials, but no DNA bands were amplified from other selected poultry disease viruses, indicating that the established PCR method has high specificity .

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Abstract

The invention discloses a primer pair for preparing a kit for detecting type-4 avian adenovirus and an application thereof. The primer pair comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown by SEQ ID No.1; and the nucleotide sequence of the downstream primer is shown by SEQ ID No.2. The invention also discloses a kit comprising the primer pair and a method for detecting the type-4 avian adenovirus. The primer pair can specifically amplify the Hexon gene segment of the type-4 avian adenovirus in a sample, the detection sensitivity is high, the lowest detection limit reaches 7.2*10<4>ng / mu L, the result is accurate, and the primer pair can be applied to the detection and identification of the type-4 avian adenovirus; the primer pair is universal in detecting different strains of the type-4 avian adenovirus; and the rapid, specific and accurate detection method of the type-4 avian adenovirus provides a detection tool for detecting and controlling the prevalence of the disease.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a pair of primers for preparing a kit for detecting avian adenovirus type 4 and an application thereof. Background technique [0002] Fowl adenovirus (Fowl adenovirus, FAdV) is a non-enveloped double-stranded DNA virus, which can be divided into three groups I, II and III, of which FAdV in group I has 12 serotypes (FAV1-12). Different serotypes of group Ⅰ adenoviruses have different pathogenicity to poultry. Rapid and accurate distinction of different serotypes of group Ⅰ adenoviruses is of great significance for clinical diagnosis and treatment. [0003] The main structural proteins encoded by avian adenovirus are hexon (Hexon), penton (matrix, IIIa), fibril, pVI, pVII, pVIII. It has been confirmed that the hexon protein has major genus and subgenus-specific epitopes and minor species-specific epitopes, which can trigger a very strong neutralization reaction. When the hexon is repl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/70C12Q1/686
CPCC12Q1/686C12Q1/701C12Q2545/113
Inventor 廖敏莫开昆周继勇郑肖娟
Owner ZHEJIANG UNIV
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