Specific primer pair for rapidly detecting cucumber green mottle mosaic viruses (CGMMV) and application thereof

A specific primer pair, green mottled flower technology, applied in the direction of microorganisms, microorganism-based methods, microorganism determination/inspection, etc., can solve the problems of yield loss, plant dwarf, fruit color spots, etc., and achieve high sensitivity and specificity. Strong, reliable results

Inactive Publication Date: 2011-08-03
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus was first discovered on cucumbers by Ainsworth in England in 1935. It can infect many cucurbit crops. Typical symptoms on cucumbers are stains, blisters and deformation on leaves, dwarfing of plants, and infected fruits are usually asymptomatic. , but some strains ca...

Method used

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  • Specific primer pair for rapidly detecting cucumber green mottle mosaic viruses (CGMMV) and application thereof
  • Specific primer pair for rapidly detecting cucumber green mottle mosaic viruses (CGMMV) and application thereof
  • Specific primer pair for rapidly detecting cucumber green mottle mosaic viruses (CGMMV) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1, the design of specific primer pair

[0021] The specific primer pair designed to assist in the identification of cucumber green mottle mosaic virus (CGMMV) is as follows:

[0022] Upstream primer: 5'-gaagagtccagttctgtttc-3' (sequence 1 of the sequence listing);

[0023] Downstream primer: 5'-accctcgaaactaagctttc-3' (Sequence 2 of the Sequence Listing).

[0024] The target sequences of the upstream primer and the downstream primer are shown in sequence 3 of the sequence listing.

Embodiment 2

[0025] Example 2, the application and specificity of specific primer pairs in assisting the identification of CGMMV

[0026] Identify various viruses (CGMMV, TMV and ORSV) respectively with the specific primer pair (upstream primer shown in sequence 1 of the sequence listing and sequence 2 shown in the sequence listing) of the specific primers designed in Example 1. The specific steps are as follows:

[0027] 1. Extract the total RNA of the virus and reverse transcribe it into cDNA.

[0028] The reverse transcription system (30 μL) contained 0.5 μL M-MLV Reverse Transcriptase (200 U / μL), 1 μL RNA and 0.5 μL downstream primers (10 mmol / L).

[0029] 2. Using the cDNA obtained in step 1 as a template, perform PCR amplification to obtain a PCR amplification product.

[0030] PCR amplification system (25μL): 18.2μL ddH 2O, 2.5μL 10×PCR buffer, 1.0μL dNTP mix (10mmol / L), 0.5μL upstream primer (10mmol / L), 0.5μL downstream primer (10mmol / L), 2μL template, 0.3μL Taq DNA polymerase (5...

Embodiment 3

[0033] Embodiment 3, the sensitivity of specific primer pair in auxiliary identification CGMMV

[0034] Cucumber green mottle mosaic virus can cause dwarfing of plants, delayed fruiting, most of the fruits turn yellow or white and produce dark green blister-like necrotic spots, loss of yield and even lead to infertility and termination of production. Cucumber green mottle mosaic virus infection on cucurbit leaves is manifested as: small yellow spots appear on new leaves, and then mosaic leaves appear with dark green protrusions, stains, blisters and deformation are caused on the leaves, and the leaf veins fade into green bands . Cucurbit leaves were inoculated with cucumber green mottle mosaic virus, and the fresh leaves showing signs of infection were taken as samples to be tested. Fresh cucurbit leaves that were not inoculated with cucumber green mottle mosaic virus and had no signs of infection were taken as control samples.

[0035] 1. Extract the total RNA of the sample...

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Abstract

The invention discloses a kit for rapidly detecting cucumber green mottle mosaic viruses (CGMMV) and application thereof. The specific primer pair provided in the invention comprises a DNA (Deoxyribonucleic Acid) shown as a sequence 1 in a sequence table and a DNA shown as a sequence 2 in the sequence table. When used for detecting CGMMV, the specific primer pair or kit has the advantages of simple and rapid operation, reliable result, strong specificity and high sensitivity and is suitable for field detection at ports. The specific primer pair or kit can be used for port detection of the CGMMV and investigation of field epidemic situations, and is especially suitable for use in each quarantine and inspection station, each port inspection and quarantine office, plant virus research institute and the like of agricultural departments.

Description

technical field [0001] The invention relates to a pair of specific primers for rapid detection of cucumber green mottle mosaic virus and its application. Background technique [0002] Cucumber Green Mosaic Mottle Virus (CGMMV), a member of the genus Tobacco Mosaic Virus, mainly infects Cucurbitaceae crops including gourds, cucumbers, watermelons, muskmelons, and muskmelons, causing serious damage to production. harm. The virus was first discovered on cucumbers by Ainsworth in England in 1935. It can infect many cucurbit crops. Typical symptoms on cucumbers are stains, blisters and deformation on leaves, dwarfing of plants, and infected fruits are usually asymptomatic. , but some strains can cause severe staining and deformation of the fruit, causing yield losses of up to 15%, producing only slight mottled and dwarfed leaves on watermelons, but severe discoloration or rot in the fruit. Some Asian strains do not show symptoms on leaves but can cause considerable yield loss. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/94
Inventor 李明福张永江丁建云李桂芬田永蕾陈笑瑜马洁赵竹魏梅生朱水芳
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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