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Absolute fluorescent quantitative PCR detection method for chicken infectious anemia viruses

A chicken infectivity and fluorescence quantitative technology, which is applied in the field of absolute fluorescence quantitative PCR detection of chicken infectious anemia virus, can solve the problem of low detection accuracy of chicken infectious anemia virus and lack of methods for quantitative detection of chicken infectious anemia virus And other issues

Pending Publication Date: 2021-03-19
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

At present, in production practice, the detection accuracy of chicken infectious anemia virus vaccine contamination residue and low content chicken infectious anemia virus in clinical samples is low, and there is also a lack of sensitive and specific quantitative detection methods for chicken infectious anemia virus

Method used

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  • Absolute fluorescent quantitative PCR detection method for chicken infectious anemia viruses
  • Absolute fluorescent quantitative PCR detection method for chicken infectious anemia viruses
  • Absolute fluorescent quantitative PCR detection method for chicken infectious anemia viruses

Examples

Experimental program
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Embodiment

[0036] (1) Extraction of Genomic DNA of Chicken Infectious Anemia Virus: Genomic DNA of Chicken Infectious Anemia Virus was extracted by using Axygen’s DNA mini-prep kit AxyPrep TM Multisource Genomic DNA complete.

[0037] (2) PCR detection of tissue samples infected with chicken infectious anemia virus: the primers are a pair of primers for the VP3 gene of chicken infectious anemia virus synthesized in the laboratory, the amplified fragment size is 366bp, and the primer sequence is as follows: upstream primer For VP3F: 5′-ATGAACGCCTCTCCAAGAAGATAC-3′; the downstream primer is VP3R: 5′-TTACAGTCTTATACGCCTTTTTGCG-3′; in this experiment, PCR amplification was performed using 2 xTaq Master Mix PCR enzyme from Nanjing Nuoweizan Biotechnology Co., Ltd., and the reaction system For 25 μL: 2 x Taq Master Mix 12.5 μL, template 2 μL, upstream primer and downstream primer 1 μL each, sterilized ddH 2 O 8.5 μL. The reaction program was: 95°C for 5 min; 95°C for 30 s, 56°C for 30 s, 72°C...

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Abstract

The invention relates to an absolute fluorescent quantitative PCR detection method for chicken infectious anemia virus. Primers for specifically amplifying a VP1 gene segment of the chicken infectiousanemia virus are scientifically designed, and then the absolute fluorescent quantitative PCR method is established by using a TB Green<TM>Premix Ex Taq<TM>II reagent of the TaKaRa Company. Accordingto a highly conserved region of a VP1 gene of the chicken infectious anemia virus, upstream and downstream primers with the amplified fragment sizes of 140 bp are scientifically designed and synthesized, and the sequences of the primers are as follows: the sequence of the upstream primer is VP1-F: 5'-GCCCCGGTACGTATAGTGTG-3', and the sequence of the downstream primer is VP1-R: 5'-CCCGTACATGTGGTCTGCAT-3'. The method can be used for rapid and specific detection of chicken infectious anemia virus vaccine contamination residues and low-content chicken infectious anemia viruses in clinical samples by enterprises; and by quantitatively detecting the content of the chicken infectious anemia virus, the change rule and the like of the chicken infectious anemia virus in vivo can be known, and an effective means is provided for further researching the molecular biological characteristics of the chicken infectious anemia virus and making an effective prevention and control strategy.

Description

technical field [0001] The invention relates to an absolute fluorescence quantitative PCR detection method for chicken infectious anemia virus, which belongs to the field of biotechnology. Background technique [0002] Chicken Infectious Anemia Virus (CIAV) infection mainly causes obstructive anemia and generalized lymphoid tissue atrophy in chicks; adult chickens often show subclinical infection after infection with Chicken Infectious Anemia Virus, leading to production performance and immunity. Functional decline. Chicken infectious anemia virus can be transmitted both horizontally and vertically. Since it was first reported in 1979, the chicken infectious anemia virus has spread throughout the world. In my country, chicken infectious anemia virus was isolated for the first time in Heilongjiang Province in 1992, and chicken infectious anemia virus was successively isolated in chicken flocks in Henan, Shandong, Jiangsu, Liaoning, Jilin and other places. In recent years, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2545/113C12Q2563/107C12Q2537/16
Inventor 叶建强张俊马莉李拓凡万志敏邵红霞秦爱建
Owner YANGZHOU UNIV
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