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Method for detecting cucumber green mottle mosaic virus based on one-step process RPA technology

A green mottled flower and leaf virus technology, applied in the field of plant quarantine, can solve the problems of cumbersome operation steps and high price

Inactive Publication Date: 2018-08-28
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PCR technology usually needs to be equipped with special equipment, which is expensive and has cumbersome operation steps.

Method used

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  • Method for detecting cucumber green mottle mosaic virus based on one-step process RPA technology
  • Method for detecting cucumber green mottle mosaic virus based on one-step process RPA technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, design and synthesis of primers

[0054] After a large number of sequence analysis, sequence design, artificial screening optimization, and effect verification, the specific primer pair for identification of cucumber green mottle mosaic virus was obtained. The specific primer pair consisted of CGMMV-RPA-F and CGMMV-RPA-R. The primer sequences are as follows:

[0055] CGMMV-RPA-F: 5'-AATGTGCGAGTTTGTTATGC-3' (SEQ ID NO: 1);

[0056] CGMMV-RPA-R: 5'-TCTTGGAAGATCGGAGGG-3' (SEQ ID NO: 2);

[0057] Among them, CGMMV-RPA-F is the forward primer, and CGMMV-RPA-R is the reverse primer.

[0058] The theoretical amplification product size of the specific primer pair is 368bp, and its nucleotide sequence is shown as sequence 3 in the sequence list.

Embodiment 2

[0059] Embodiment 2, establishment of method

[0060] One, the method for identifying whether the virus to be tested is cucumber green mottle mosaic virus

[0061] 1. Extract the total RNA of the virus to be tested.

[0062] 2. Using the total RNA extracted in step 1 as a template, use the primer pair designed in Example 1 to utilize Liquid Basic RT for one-step RPA amplification.

[0063] The reaction system is as follows (50μl): primer CGMMV-RPA-F 2.4ul (10μmol / L), primer CGMMV-RPA-R2.4ul (10μmol / L), Rehydration buffer 29.5μl, template 200ng, and make up the rest with sterile water 23.75ul; finally add 2.5ul MgAC (280mmol / L). Among them, both Rehydration buffer and MgAC are derived from Liquid Basic RT.

[0064] The reaction conditions are as follows: react at 42° C. for 45 minutes.

[0065] 3. Take the product of step 2 and detect it by agarose gel electrophoresis. And the leaves of plants infected with CGMMV were used as positive control samples.

[0066] If the ...

Embodiment 3

[0076] Embodiment 3, specificity experiment

[0077] The tested viruses were as follows: Cucumber green mottle mosaic virus (CGMMV), cucumber mosaic virus (CMV), Arabis mosaic virus (ArMV) and tomato ringspot virus ( Tomato ringspot virus, ToRSV).

[0078] Detection was carried out according to the method of Step 1 of Example 2.

[0079] see results figure 1 . figure 1 Among them, 1 corresponds to the positive control sample containing CMV, 2 corresponds to the Beijing melon sample containing CMV, 3 corresponds to the Liaoning zucchini sample containing CMV, and 4 corresponds to Shanxi melon samples to be tested containing cucumber green mottle mosaic virus, 5 corresponds to tobacco samples containing cucumber mosaic virus, 6 corresponds to tobacco samples containing A. thaliana mosaic virus, and 7 corresponds to tobacco samples containing tomato ringspot virus. The positive control sample containing CMV and the 3 samples to be tested were all positive, while the samples c...

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Abstract

The invention discloses a method for detecting a cucumber green mottle mosaic virus based on a one-step process RPA technology. The method is characterized in that a primer pair for detecting the cucumber green mottle mosaic virus is provided, wherein the primer pair is composed of a primer CGMMV-RPA-F and a primer CGMMV-RPA-R. The primer pair is used for detecting or detecting whether the cucumber green mottle mosaic virus or a to-be-detected plant sample is infected with the cucumber green mottle mosaic virus or not in an auxiliary mode. The primer pair provided by the invention has the advantages of good specificity and high sensitivity. The detecting method has the advantages of simple and convenient operation, high detecting efficiency and the like, and can meet basic needs of port and field detection.

Description

technical field [0001] The invention belongs to the technical field of plant quarantine and relates to biological detection technology, in particular to a pair of one-step RPA primers for detecting cucumber green mottle mosaic virus and its use in identifying cucumber green mottle mosaic virus or detecting whether plant materials are infected with cucumber green mottle mosaic Application in mosaic virus. Background technique [0002] Cucumber green mottle mosaic virus (CGMMV) belongs to the Tobamovirus genus of the family Tymoviridae and is an important quarantine pest in my country. Serious threat to Cucurbitaceae crops, is one of the important viruses infecting Cucurbitaceae crops. Currently, CGMMV is distributed in more than 30 countries and regions around the world. In 2002, my country's quarantine department discovered the virus for the first time in seedlings imported from Japan. Subsequently, many cases of imported CGMMV occurred in Liaoning, Beijing, Zhejiang, Heb...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/94
CPCC12Q1/6844C12Q1/701C12Q2521/507C12Q2522/101
Inventor 邱艳红王超楠张永江朱水芳
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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