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Avian reovirus TaqMan MGB probe real-time fluorescent quantitative PCR detection kit and application thereof

An avian reovirus and kit technology, which can be used in the determination/inspection of microorganisms, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problems of difficulty in ARV prevention and control, and achieve good specificity.

Pending Publication Date: 2022-07-29
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the ARV virus has a certain resistance to the external environment, so it is very difficult to prevent and control ARV in the modern high-density breeding mode
There is no report on the detection of ARV virus using the TaqMan minor groove binder (MGB) probe method

Method used

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  • Avian reovirus TaqMan MGB probe real-time fluorescent quantitative PCR detection kit and application thereof
  • Avian reovirus TaqMan MGB probe real-time fluorescent quantitative PCR detection kit and application thereof
  • Avian reovirus TaqMan MGB probe real-time fluorescent quantitative PCR detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Design of primers and probes for detection of avian reoviruses and establishment of methods

[0051] 1. Design and synthesis of primers and probes

[0052] According to the sequence of the conserved region of the ARV S1 gene in the NCBI database, a pair of RT-PCR primers (named ARV-S1F and ARV-S1R) and a MGB probe (named as ARV-S1P).

[0053] And according to the sequence of ARV S1 gene, the primers (named ARV-F and ARV-R) for constructing plasmid standard were designed. Primers and probes were synthesized by ThermoFisher Company, and the sequences of primers and probes are shown in Table 1.

[0054] Table 1 shows primer and probe sequences

[0055]

[0056]

[0057] In the above, the 5' end of the ARV-S1P probe is labeled with a FAM group, and the 3' end of the probe is labeled with an MGB group.

[0058] 2. Preparation of standard products

[0059] Using the cDNA extracted and reverse transcribed from avian reovirus ARV S1133 strain as a template...

Embodiment 2

[0078] Example 2. Detection of Avian reovirus (ARV)

[0079] A specific experiment

[0080] 1. Nucleic acid acquisition

[0081] The nucleic acids of different strains of ARV and common pathogens of avian origin were extracted as templates.

[0082] ARV S1133 virulent strain, ARV S1133 attenuated strain, ARV Guangxi isolate GuangxiR1 strain, avian influenza virus (AIV), chicken parvovirus (ChPV), avian adenovirus (FAdV), Newcastle disease virus (NDV) and chicken infectious anemia (CIAV).

[0083] 2. Carry out RT-PCR amplification according to the method of 4 in Example 1.

[0084] The result is as figure 1 As shown, the detection results of ARV TaqMan MGB probe RT-PCR detection method for ARV S1133 virulent strain, ARVS1133 attenuated strain and ARV Guangxi isolate GuangxiR1 strain all showed typical positive reactions, and a specific S-type fluorescence amplification curve appeared. , the CT values ​​were 25.2, 31.31 and 21.12, respectively. The test results for avian i...

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Abstract

The invention discloses an avian reovirus TaqMan MGB probe real-time fluorescent quantitative PCR (polymerase chain reaction) detection kit and application thereof. The invention provides a primer probe set which is composed of a primer 1, a primer 2 and a probe. The nucleotide sequence of the primer 1 is sequence 1; the nucleotide sequence of the primer 2 is a sequence 2; and the nucleotide sequence of the probe is sequence 3. A TaqMan MGB probe technology is utilized to establish a real-time fluorescent quantitative PCR technology for specifically detecting the ARV virus, and the real-time fluorescent quantitative PCR technology is successfully applied to rapid detection and diagnosis of the ARV virus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a real-time fluorescent quantitative PCR detection kit for avian reovirus TaqMan MGB probe and its application. Background technique [0002] Avian reovirus (ARV) is a member of the genus Orthoreovirus of the family Reovirdase. In 1954, Fahey and Crawley first isolated ARV from the respiratory tract of chickens with chronic respiratory disease. Since the 1880s in my country, reports of the disease have appeared all over the country. At present, ARV infection has a global distribution, and ARV mainly infects chickens, turkeys and some birds. The clinical symptoms of ARV infection are mainly viral arthritis syndrome. Affected chickens often have lameness. The infection can also cause damage to cells in the liver, pancreas, thymus, bursa and spleen of chickens, resulting in severe immunosuppression. . ARV infection will cause the overall production performance of chickens to decline, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2561/101C12Q2563/107C12Q2545/113C12Q2531/113
Inventor 谢芝勋王盛万丽军任红玉谢丽基谢志勤邓显文范晴罗思思张艳芳黄娇玲曾婷婷张民秀
Owner GUANGXI VETERINARY RES INST
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