Fluorescence quantitative RT-PCR detection reagent for West Nile virus and preparation method and use thereof

A West Nile virus and RT-PCR technology, which is used in the preparation of reagents and the field of reagents for West Nile virus detection, achieves the effect of wide application range, high throughput and strong sensitivity of samples

Inactive Publication Date: 2006-10-04
CHECKOUT & QUARANTINE TECH CENT YUNNAN ENTRY &EXIT CHECKOUT & QUARANTINE BUR
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Problems solved by technology

RT-nested PCR can reliably detect WNV nucleotides from horse brain tissue, however, non-specific contamination often leads to false-positive results in RT-nested PCR

Method used

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  • Fluorescence quantitative RT-PCR detection reagent for West Nile virus and preparation method and use thereof

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Embodiment Construction

[0028] 1. Construction of WNV E gene recombinant plasmid

[0029] According to "Molecular Cloning Experiment Guide" (Third Edition) translated by Huang Peitang et al., China Science Press, January 2003, pages 1217 to 1259, the West Nile virus E gene was cloned and sequenced, and the WNVE gene recombinant plasmid was constructed. , and the recombinant plasmid was named pBAD-E.

[0030] 2. Design primers and probes

[0031]The present invention selects the conserved segment of the WNV E gene as the target, and compares the homology analysis and comparison between the DNA sequences of the virus genes of the WNV epidemic strains reported in GenBank and the DNA sequences of the West Nile virus WNV genes cloned and sequenced in 1 above. , select the conserved fragment (71bp) of WNV gene, apply primerExpress software and primer prere5.0 software, design primers and probes, the synthesis of primers and probes adopts β-acetonitrile phosphite chemical synthesis method, and uses automat...

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Abstract

The related bio-reagent is designed by: with conservative West Nile virus E 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 71bp.

Description

technical field [0001] The invention relates to a biological agent designed and synthesized by targeting West Nile virus gene fragments, in particular to a reagent capable of detecting West Nile virus, and a preparation method and application of the reagent. Background technique [0002] West Nile virus (WNV) disease is a severe zoonotic infectious disease. The pathogen of the disease belongs to the Japanese encephalitis virus serogroup of the Flavivirus genus with Japanese encephalitis virus, Molle Valley encephalitis virus, and St. Louis encephalitis virus, and they have crossover in serological reactions. Diseases caused by viruses of the Flaviviridae family are capable of causing serious public health problems. WNV can be transmitted by blood-sucking vectors such as mosquitoes or ticks, and most of them lack effective treatment and preventive measures, which poses a severe challenge to disease control. In the past 20 years, West Nile virus outbreaks among humans and an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
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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