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RT-LAMP (loop-mediated isothermal amplification) rapid detection kit for cucumber green mottle mosaic virus (CGMMV) and detection method

A technology of RT-LAMP and detection kit, which is applied in the field of agricultural science to achieve the effect of accurate early diagnosis, high accuracy and large amount of amplification products

Inactive Publication Date: 2014-08-13
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, RT-LAMP technology has not yet been applied to the detection of cucumber green mottle mosaic virus (CGMMV)

Method used

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  • RT-LAMP (loop-mediated isothermal amplification) rapid detection kit for cucumber green mottle mosaic virus (CGMMV) and detection method
  • RT-LAMP (loop-mediated isothermal amplification) rapid detection kit for cucumber green mottle mosaic virus (CGMMV) and detection method
  • RT-LAMP (loop-mediated isothermal amplification) rapid detection kit for cucumber green mottle mosaic virus (CGMMV) and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Optimization of RT-LAMP reaction conditions for cucumber green mottled mosaic virus

[0069] ①Design of RT-LAMP primers for cucumber green mottle mosaic virus

[0070] Cucumber green mottle mosaic virus reported by NCBI

[0071] (http: / / www.ncbi.nlm.nih.gov / nuccore / NC_001

[0072] 801.1) The complete nucleotide genome sequence, after analysis by the software DNAstar, find out the nucleotide coat protein region 5763~6248bp of the virus, use the corresponding region 5763~6248bp on NP_044580.1 as the template reference sequence, and use the online LAMP primer design software Primers were designed with Primer Explorer V4 (http: / / primerexplorer.jp / elamp4.0.0 / index.html). Then compare and select the designed multiple pairs of primers according to the corresponding 3' or 5' end stability and primer dimer parameters provided by the software, and finally select a set of the most suitable primers. The sequence is as follows:

[0073] F3 CCGTCAGGACTTTACTTA

[0074] B...

Embodiment 2

[0095] Example 2, the specificity and visualization test of the RT-LAMP reaction for crude extraction of cucumber green mottle mosaic virus sample

[0096] ①Use the rapidly extracted samples to crudely extract total RNA for RT-LAMP

[0097] The method of rapid crude RNA extraction can enable CGMMV RT-LAMP to quickly detect disease samples and improve efficiency. The method of quickly extracting RNA is: use 0.1g of the diseased leaf just collected, grind it in 400μL of 0.5M NaOH solution, take 10μL of the grinding solution and add it to 490μL Tris-HCL (pH8.0), and directly take 0.8μL of the liquid as a crude Extract the total RNA template for CGMMV RT-LAMP detection experiment ( Figure 5 ).

[0098] ② Specificity test of cucumber green mottle mosaic virus RT-LAMP

[0099] In order to verify the specificity of the RT-LAMP method, the virus that can infect melons was selected as the control cucumber mosaic virus (CMV) and the tobacco mosaic virus (TMV) as the control. The to...

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Abstract

The invention discloses an RT-LAMP (loop-mediated isothermal amplification) rapid detection kit for cucumber green mottle mosaic virus (CGMMV) and a detection method. The method comprises the steps of extracting plant viruses RNA, detecting an RT-LAMP reaction and electrophoresis detection or fluorochrome, and determining whether a plant carries the viruses. An RT-LAMP primer is designed according to a coat protein gene conserved region of CGMMV, and an RT-LAMP technology is adopted to establish a detection technology which can rapidly and sensitively detect the cucurbitaceous plant CGMMV such as cucumbers, watermelons and calabashes with high specificity and an application method thereof in diagnosing disease. The detection method provided by the invention can utilize the rapid extraction RNA and conventional RNA extraction as an RNA template for an RT-LAMP experiment. The method is utilized to rapidly identify a CGMMV plant sample, the targeted quarantine inspection protective measure is adopted, and the loss caused by the disease is reduced.

Description

technical field [0001] The invention belongs to the field of agricultural science and technology, and relates to a rapid detection technology of cucumber green mottle mosaic virus on Cucurbitaceous plants such as watermelon, cucumber and gourd and its application in disease diagnosis, in particular to a RT of cucumber green mottle mosaic virus -LAMP rapid detection kit and detection method. Background technique [0002] Cucumber green mottle mosaic virus (CGMMV) is a member of the family Tombusviridae and the genus Tobamovirus. The disease seriously damages Cucurbitaceae crops and is a quarantine pest in my country. Cucumber green mottle mosaic virus infected seedlings at the seedling stage have bright green or dark green mottled 2-3 leaves at the top of the seedlings. The leaves are relatively flat and dark green mottled diseased parts are raised. It becomes smaller, causing the plants to dwarf, and the leaves are mottled and twisted, showing a systemic infection. When t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
Inventor 陶小荣李靖玉卫其巍张雯娜吴鉴艳
Owner NANJING AGRICULTURAL UNIVERSITY
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