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

A PPR and fluorescence quantitative technology, applied in biochemical equipment and methods, fluorescence/phosphorescence, microbial measurement/inspection, etc., can solve the problem of low sensitivity and specificity of detection methods, inability to make rapid diagnosis, and virus isolation Time-consuming methods and other problems, to achieve the effect of wide application range of samples, safe use, and intuitive results

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

[0008] For the rapid detection of PPR virus, the virus isolation method is time-consuming, cannot make a rapid diagnosis, and has potential biological safety hazards. Sensitive hosts or cells need to be selected
Sensitivity and specificity of other detection methods are not high

Method used

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

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Experimental program
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Effect test

Embodiment Construction

[0032] 1. Construction of PPV H gene recombinant plasmid

[0033] According to "Molecular Cloning Experiment Guide" (Third Edition) translated by Huang Peitang et al., China Science Press, January 2003, page 1217 to page 1259, clone and sequence the H gene of Peste des petits ruminants virus, and construct PPV H gene recombination Plasmid, the recombinant plasmid was named pBAD-H.

[0034] 2. Design primers and probes

[0035] The present invention selects the conserved fragment of the PPV H gene as the target, and compares the homology analysis and comparison between the DNA sequences of the virus genes of the PPV isolates reported in GenBank and the PPV gene DNA sequences of the above-mentioned cloned and sequenced Peste des petits ruminants. Select the conserved fragment (69bp) of the PPV gene, and use primerExpress software and primer prere5.0 software to design primers and probes. The synthesis of primers and probes adopts the chemical synthesis method of β-acetonitrile ph...

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Abstract

The related bio-reagent is designed by: with conservative PPR (PESTE DES PETITS RUMINANTS), virus H 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 76bp.

Description

technical field [0001] The invention relates to a biological agent designed, synthesized and prepared with the gene fragment of Peste des petits ruminants virus as the target, especially a reagent capable of detecting Peste des pets ruminants virus, a preparation method and application of the reagent. Background technique [0002] Peste des petits ruminants (PPR) is a class A severe infectious disease stipulated by the FAO / OIE. my country defines it as a class I animal disease. Goats are highly susceptible, but other wild animals can also occur. The disease is characterized by sudden onset of fever, depression, ocular and nasal discharge, mouth sores, respiratory disturbances, coughing, foul-smelling diarrhea, and high mortality. The incidence rate of this disease can reach 100%, and the mortality rate in severe outbreak period is 100%. . [0003] The disease was first discovered in Côte d'Ivoire, West Africa in 1942, and subsequently confirmed to exist in Nigeria, Senegal...

Claims

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Application Information

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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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