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Method for rapidly detecting various viruses of melons

A technology of cucumber mosaic virus and pumpkin mosaic virus, which is applied in the field of plant virology and can solve the problems of consuming more time and reagents

Inactive Publication Date: 2012-06-06
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Single-plex RT-PCR (sRT-PCR) and nucleic acid hybridization detection methods can only detect one virus in one reaction, and most of the field watermelon viruses are compound infections, so it takes more time and reagents to detect with these two methods

Method used

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  • Method for rapidly detecting various viruses of melons
  • Method for rapidly detecting various viruses of melons
  • Method for rapidly detecting various viruses of melons

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Single-plex RT-PCR detection of 5 kinds of single melon virus samples in Yangling, Shaanxi

[0054] Six samples were taken: melon leaves mixed with ZYMV, WMV, TMV, SqMV, CMV and 5 kinds of viruses, and the sample numbers were: sample 1, sample 2, sample 3, sample 4, sample 5, sample 6.

[0055] Extraction of total viral RNA

[0056] Weigh 0.5g of melon leaves mixed with ZYMV, WMV, TMV, SqMV, CMV and 5 kinds of viruses (marked as sample 2, sample 3, sample 4, sample 5, sample 6 respectively) and put them in deep freeze at -80°C Add liquid nitrogen to the mortar and grind thoroughly, quickly transfer to RNase-free sterile 1.5mLeppendorf tube, add 500μL phenol / chloroform (1:1) and 500μL RNA extraction buffer (Tris-HCl20mmol / L, SDS1%, NaCl 200mmol / L, EDTA50mmol / L), shake fully for 2min, centrifuge at 12,000g for 5min at 4°C. The supernatant was extracted once more with an equal volume of phenol / chloroform, and centrifuged at 12,000 g for 5 min at 4°C. Add an eq...

Embodiment 2

[0063] Example 2 Multiplex RT-PCR detection of muskmelon virus samples infected by multiple viruses in Yangling, Shaanxi

[0064] Extraction of total viral RNA

[0065] The melon mosaic samples (marked as sample 1, sample 2, sample 3, sample 4, sample 5, and sample 6) from six places in Shaanxi, Yangling, Hancheng, Liquan, Fufeng, Zhouzhi, and Wugong, respectively, were 0.5 g was placed in a deep-frozen mortar at -80°C with liquid nitrogen and thoroughly ground, quickly transferred to an RNase-free sterile 1.5 mL Eppendorf tube, and 500 μL phenol / chloroform (1:1) and 500 μL RNA extraction buffer (Tris- HCl 20mmol / L, SDS 1%, NaCl 200mmol / L, EDTA 50mmol / L), shake well for 2min, centrifuge at 12,000g for 5min at 4°C. The supernatant was extracted once more with an equal volume of phenol / chloroform, and centrifuged at 12,000 g for 5 min at 4°C. Add an equal volume of 4M LiCl to the supernatant, precipitate at 4°C for more than 4 hours, and centrifuge at 12,000g for 15 minutes at...

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Abstract

The invention also provides a method for rapidly detecting various viruses of melons. The method comprises the following steps of: extracting total RNA of a virus; performing reverse transcription to obtain a cDNA template; performing multiple polymerase chain reaction PCR on the template by using primers; detecting the product; and determining the type of the virus, wherein 5 pairs of primers from SEQ ID No. 1 to 10 are applied in the multiple PCR. The method has the advantages of saving the time, reducing the cost and improving the efficiency during the detection of the complex infective virus of the melons.

Description

technical field [0001] The invention belongs to the technical field of plant virology, in particular to a method for rapidly detecting various melon viruses. Background technique [0002] my country is one of the main producers of watermelons, with an average annual watermelon planting area of ​​over 18 million mu, ranking first in the world. Among them, Northwest China is an important high-quality watermelon producing area in my country. Virus disease is a common and very harmful disease in watermelon production. The incidence rate is 10%-30% in general years, and it can be as high as 50%-80% in re-emergence years. The large-scale occurrence of watermelon virus disease has caused a large amount of watermelon production reduction and quality decline, which will seriously affect the high yield, stable yield, high quality, high benefit and production enthusiasm of watermelon farmers. [0003] There are mainly five kinds of viruses infecting watermelon: cucumber mosaic virus ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 吴云锋
Owner NORTHWEST A & F UNIV
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