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RT-qPCR kit capable of detecting three citrus viruses synchronously

A simultaneous detection and kit technology, applied in the field of plant virus diagnosis, can solve the problems of contact with toxic substances, high time cost, high probability of pollution, etc., and achieve good application prospects and easy operation

Inactive Publication Date: 2016-04-13
SOUTHWEST UNIVERSITY
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Problems solved by technology

[0004] The object of the present invention is to provide a kind of RT-qPCR reagent kit that detects three kinds of citrus viruses synchronously, aim at solving biological detection method limited by environmental conditions, time-consuming and laborious; Serological method sensitivity and accuracy are low; Molecular biology method There are cumbersome operations that require multiple virus divisions, high time costs, high chances of contamination, and the need to contact toxic substances; and further improve detection sensitivity

Method used

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  • RT-qPCR kit capable of detecting three citrus viruses synchronously
  • RT-qPCR kit capable of detecting three citrus viruses synchronously
  • RT-qPCR kit capable of detecting three citrus viruses synchronously

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Embodiment 1

[0089] Embodiment 1, real-time fluorescent RT-qPCR detection and quantitative examples of CYVCV, CTLV and CTV in the present invention

[0090] 1. Extraction of total nucleic acid from the sample: use a sterile blade to take 0.1 mg of the midrib of citrus leaves mixed with CYVCV, CTLV and CTV and place it in a 1.5ml centrifuge tube, use the nucleic acid extraction kit MiniBESTViralRNA / DNAExtractionKitVer.4.0 (TAKARA) Product Instructions Extract viral nucleic acid from sample leaves.

[0091] 2. Multiplex fluorescent quantitative RT-qPCR: prepare real-time fluorescent RT-qPCR reaction mixture on ice in the dark, including 12.5 μl of RT-qPCR reaction solution, 0.3 μl of RT-Taq enzyme, and 1.5 μl of specific primer mixture for CYVCV, CTV and CTLV , plus ddH 2 0 until the total volume of the reaction system is 23 μl; take 2 μl each of the positive control substance, the negative control substance, and the above-mentioned total nucleic acid to be tested into RT-qPCR tubes, add 23...

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Abstract

The invention discloses a realtime fluorescent quantitative RT-qPCR kit capable of detecting CYVCV, CTV and CTLV synchronously. The RT-qPCR kit comprises three pairs of virus-specific RT-qPCR primers which are shown as in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. By using the RT-qPCR kit, synchronous detection and virus quantitation of CYVCV, CTV and CTLV can be realized through a RT-qPCR reaction. Compared with conventional RT-PCR detection methods, a method using the RT-qPCR kit has the advantages that sensitivity in detecting CYVCV, CTV and CTLV is improved by higher than 100 times, detection time is saved higher than 3 times, and pollution probability is lowered higher than 30%. The RT-qPCR kit has a good application prospect in diagnosing disease occurrence, monitoring virus prevalence and maintaining safety of citrus industry.

Description

technical field [0001] The invention belongs to the technical field of plant virus diagnosis, in particular to an RT-qPCR kit for synchronously detecting three citrus viruses of CYVCV, CTV and CTLV. Background technique [0002] Citrus is the world's number one fruit and the fifth most traded agricultural product globally. In 2013, my country's citrus cultivation area was 36.33 million mu, with an output of 33.21 million tons, ranking first in the world. In recent years, with the rapid development of the citrus industry, multi-pathogen mixed infection and secondary infection of citrus have become more and more common. Citrus Yellow Vein Virus, Citrus Decay Virus and Citrus Broken Leaf Virus are widespread in production and cause great damage, and there is a mixed infection situation, which poses a threat to the safety of my country's citrus industry. Citrus yellow vein disease caused by citrus yellow vein bright virus is a recent disease. When lemons and limes were infect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6851C12Q1/701C12Q2600/16C12Q2537/143C12Q2545/113
Inventor 宋震刘科宏陈洪明李中安周常勇
Owner SOUTHWEST UNIVERSITY
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