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Fluorescent quantitative RT-PCR detection reagent for wheat streak mosaic virus, as well as preparation method and application

A mosaic virus and fluorescence quantitative technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of low detection efficiency and no fluorescent signal, and achieve large detection amount, good repeatability, and wide application range wide effect

Inactive Publication Date: 2009-04-15
NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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Problems solved by technology

[0006] The Tm value of ordinary TaqMan probes is 65-72°C, and the fragment length is 25-40 nt. Due to its high specificity, if the probe does not completely match the target gene sequence, the detection efficiency will be low or even no fluorescent signal will occur. problems such as

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  • Fluorescent quantitative RT-PCR detection reagent for wheat streak mosaic virus, as well as preparation method and application

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Embodiment Construction

[0030] Hereinafter, the present invention will be further described in detail with reference to the embodiments.

[0031] 1. Design primers and probes

[0032] The present invention selects the conservative fragment of WSMV coat protein coding gene as the target, and compares the CP coding gene sequence of each strain of WSMV reported in GenBank for homology analysis and comparison, selects the conservative fragment (63bp) of the characteristic gene of WSMV, and applies primer Express 3.0 software, design primers and probes. The synthesis of primers and probes adopts the β-acetonitrile phosphamidite chemical synthesis method, and uses an automatic DNA synthesizer to synthesize OligoDNA probes. The synthesis is carried out with fluorescent labeling at both ends. The fluorescent reporter group labeled at the 5′ end of the probe is In FAM, the 3'end is labeled with a non-fluorescent quenching group and has an MGB molecule. After designing and synthesizing multiple pairs of primers an...

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Abstract

The invention provides a fluorescence quantitative RT-PCR detecting reagent for a wheat streak mosaic and a preparation method and applications thereof; the fluorescence quantitative RT-PCR detecting reagent for the wheat streak mosaic is finely designed and composed by taking the specific and more conservative CP coding gene section between each strain of the wheat streak mosaic as a target sequence; the fluorescence quantitative RT-PCR detecting reagent for the wheat streak mosaic comprises a pair of specific primers and a specific fluorescence probe; the length of a target augmentation section is 63bp; the sequences of the primers and the probe are as follows: the primer WSMV TF is 5'- agctatttgcacaggcacga-3'; the primer WSMV TR is 5'- ctgcatcatgacgtgagttgtc-3' and the probe is 5'- FAM-tgagtgcgggtactaatga-MGB-3'. The invention overcomes the defects of time waste, easy pollution and needing electrophoresis detection after augmentation of the normal RT-PCR, can carry out quick and accurate qualitative and quantitative detection on the WSMV in samples and has the advantages of simple and easy operation, intuitionistic result, high sensibility, good repetitiveness, and the like.

Description

Technical field [0001] The invention relates to a biological preparation for detecting wheat streak mosaic virus, in particular to a fluorescent quantitative RT-PCR detection reagent for wheat streak mosaic virus, and a preparation method and application thereof. Background technique [0002] Wheat streak mosaic virus (WSMV) infects almost all wheat varieties, as well as oats, barley, some corn varieties and millet, as well as many monocotyledonous weeds. The yield loss of wheat infected by WSMV is generally 30% to 50%, and severe grains can cause no harvest. The vector for transmission of wheat streak mosaic virus is the millet mite, which can transmit the virus by friction with the sap, but the seeds, soil and other insects do not transmit the virus. At present, the virus is mainly distributed in the United States, Canada, Jordan, Romania, Russia and other countries. It is a quarantine pest announced by my country. [0003] Wheat Streak Mosaic Virus belongs to the genus Wheat M...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 闻伟刚陈先锋杨文潮
Owner NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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