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Fluorescence quantitative RT-PCR detection reagent for African horse sickness virus and preparation method and use thereof

A RT-PCR, fluorescence quantitative technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of time-consuming virus isolation methods, inability to make rapid diagnosis, etc., to achieve no biological safety hazards, sample application scope Broad, high-throughput effects

Inactive Publication Date: 2008-03-26
CHECKOUT & QUARANTINE TECH CENT YUNNAN ENTRY &EXIT CHECKOUT & QUARANTINE BUR
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Problems solved by technology

[0005] Virus isolation methods are time-consuming, do not allow rapid diagnosis, and require selection of sensitive hosts or cells

Method used

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  • Fluorescence quantitative RT-PCR detection reagent for African horse sickness virus and preparation method and use thereof

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Experimental program
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Effect test

Embodiment Construction

[0027] 1. Construction of AHSV VP7 Gene Recombination Plasmid

[0028] According to "Molecular Cloning Experiment Guide" (Third Edition) translated by Huang Peitang et al., China Science Press, January 2003, page 1217 to page 1259, African horse sickness virus VP7 gene was cloned and sequenced, and AHSV VP7 gene recombination was constructed Plasmid, the recombinant plasmid was named pBAD-VP7.

[0029] 2. Design primers and probes

[0030] The present invention selects the conserved fragment of the AHSV VP7 gene as the target, and the DNA sequence of the highly conserved gene fragment VP7 gene among the 9 serotypes of AHSV reported in GenBank and the DNA sequence of the African horse sickness virus AHSV gene cloned and sequenced in the above 1 Carried out homology analysis and comparison, selected the conserved fragment (69bp) of AHSV VP7 gene, designed primers and probes according to the characteristics of VP7 gene and the principle of primer and probe design, the synthesis ...

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Abstract

The related bio-reagent is designed by: with conservative African horse sickness virus VP7 gene fragment as target object, designing and composing carefully for large quantities of primers and probes, taking optimal pairing screen test on different conditions to obtain the most proper primer and probe. The fluorescence quantitative RT-PCR testing reagent includes one couple of specific primers and one specific fluorescent probe, and the amplification fragment length is 69bp.

Description

technical field [0001] The invention relates to a biological preparation designed and synthesized by taking African horse sickness virus gene fragments as a target, especially a reagent capable of detecting African horse sickness virus, and a preparation method and application of the reagent. Background technique [0002] African Horse Sickness (AHS) is an acute or subacute viral vector-borne disease that is transmitted by insects and mainly infects horses and other equines. The disease mainly occurred in Africa, and later spread throughout the Middle East and invaded parts of Asia. It is characterized by fever, subcutaneous connective tissue and pulmonary edema, and visceral hemorrhage, and the main form is endemic and seasonal (spring and summer rainy seasons). Its harm mainly causes the death of horses and mules, and the death rate of sick horses in the new epidemic area is as high as 95%. The International Office of Epizootics (OIE) identified the disease as a category...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 花群义周晓黎董俊杨云庆徐自忠肖荣海尹尚莲
Owner CHECKOUT & QUARANTINE TECH CENT YUNNAN ENTRY &EXIT CHECKOUT & QUARANTINE BUR
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