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Primer set and kit for quickly detecting various melon viruses

A primer set and kit technology, applied in the field of plant virology, can solve problems such as time-consuming and reagent-consuming

Inactive Publication Date: 2010-12-08
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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  • Primer set and kit for quickly detecting various melon viruses
  • Primer set and kit for quickly detecting various melon viruses
  • Primer set and kit for quickly detecting various melon viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Single-plex RT-PCR detection of 5 kinds of single melon virus samples in Yangling, Shaanxi

[0051] 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.

[0052] Extraction of total viral RNA

[0053] 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, 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 eq...

Embodiment 2

[0060] Example 2 Multiplex RT-PCR detection of muskmelon virus samples infected by multiple viruses in Yangling, Shaanxi

[0061] Extraction of total viral RNA

[0062] 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 relates to a primer set and a kit for quickly detecting various melon viruses. By applying specificity primer pairs SEQ ID NO.1-10 designed according to nucleotide conserved region sequences of 5 viruses, a multiple RT-PCR system is optimized in aspects such as primer concentration, Mg2+ concentration, Taq DNA polymerase concentration, dNTPs concentration, annealing temperature and the like which influence multiple RT-PCR (M-PCR) amplification, and the primer set and a kit capable of simultaneously detecting field samples complexly infected by five viruses CMV, SqMV, TMV, WMV and ZYMV from watermelon leaf tissues are established. The primer set and the kit for detecting complex infectious viruses of melons have the advantages of saving time, reducing cost and improving efficiency.

Description

technical field [0001] The invention belongs to the technical field of plant virology, in particular to a primer set and a kit 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...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 吴云锋
Owner NORTHWEST A & F UNIV