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RNA-RP RT-PCR method capable of fast detecting melon viruses

A technology for melons and viruses, applied in the directions of DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., to achieve the effect of saving cost and time, and simple operation

Active Publication Date: 2020-04-03
ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the direct release and amplification of plant RNA, although no commercial products have been developed, reports have shown that some surfactants (such as Triton series) have certain potential in the release of plant RNA

Method used

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  • RNA-RP RT-PCR method capable of fast detecting melon viruses
  • RNA-RP RT-PCR method capable of fast detecting melon viruses
  • RNA-RP RT-PCR method capable of fast detecting melon viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The screening of solution used in the quick release of embodiment 1 plant total RNA

[0051] Weighed 8 copies of 0.05 g of CGMMV melon diseased leaves, put them in 2 mL centrifuge tubes, and crushed them with liquid nitrogen at low temperature using a tissue disruptor, and added 1 mL of RNase-free ddH2O, 1×PBS, PBST, PBST+2%PVP, 0.5% Triton X-100 aqueous solution, 0.5% Triton X-100 PBS solution, 0.5% Triton X-405 aqueous solution and 0.5% Triton X-405 PBS solution, shake and mix vigorously, 8000 rpm Centrifuge for 5 min, absorb the supernatant for later use;

[0052] Use the CGMMV-specific primer sequence CG-3467F / CG-5294R to carry out virus detection on the prepared supernatant, in which 2×1 Step Buffer (Dye PLus) 10 μL, PrimeScript 1 Step Enzyme Mix 0.8 μL, CG-3467F (10 μM) 1 μL, CG-5294R (10 μM) 1 μL, supernatant 0.5 μL, rehydration 6.7 μL, total PCR system 20 μL; 50°C for 30 min, 94°C for 2 min, 94°C for 30 sec, 57°C for 30 sec, 72°C 2 min, run 35 cycles, total ex...

experiment example 2

[0055] Experimental example 2 RNA-RP RT-PCR detection of CGMMV in tobacco and cucumber

[0056] Weigh two copies of 0.05 g of CGMMV tobacco leaves and cucumber diseased leaves, put them in 2 mL centrifuge tubes, crush them with liquid nitrogen at low temperature with a tissue breaker, and add 1 mL of PBST+2% to each tube sample PVP and 0.5% Triton X-405PBS solution, shake vigorously, mix well, centrifuge at 8000 rpm for 5 min, absorb the supernatant, and use the CGMMV-specific primer sequence CG-3467F / CG-5294R to carry out virus detection on the prepared supernatant, wherein 2×1 Step Buffer (Dye PLus) 10 μL, PrimeScript 1 Step Enzyme Mix 0.8 μL, CG-3467F (10 μM) 1 μL, CG-5294R (10 μM) 1 μL, supernatant 0.5 μL, water 6.7 μL, total A total of 20 μL of PCR system; 35 cycles of 30 min at 50°C, 2 min at 94°C, 30 sec at 94°C, 30 sec at 57°C, 2 min at 72°C, and a total extension of 2 min at 72°C;

[0057] 4 μL of PCR products were taken for electrophoresis detection, and the results...

experiment example 3

[0058] Experimental example 3 RNA-RP RT-PCR detection of CMV in tobacco and melon

[0059] Weigh 0.05 g of CMV benthamiana benthamiana and melon diseased leaves respectively, put them in 2mL centrifuge tubes, crush them with liquid nitrogen at low temperature with a tissue breaker, and add 1 mL of PBST+2%PVP to each tube sample and 0.5% Triton X-405PBS solution, shake vigorously, mix well, centrifuge at 7000 rpm for 8min, absorb the supernatant, and use specific primers CMV-R1-1F / CMV-R1-1627R combined with a one-step RT-PCR method to detect CMV, Concrete step is the same as embodiment 1, is not repeating,

[0060] 3 μL of PCR products were taken for electrophoresis detection. The results are shown in 5, indicating that both PBST+2%PVP and 0.5% Triton X-405PBS solutions can effectively release the total RNA in Nicotiana benthamiana and melon leaves for the detection of CMV.

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Abstract

The invention relates to an RNA-RP RT-PCR method capable of fast detecting melon viruses. Leaves of melon plants infected with viruses are homogenized by adopting a PBST plus a 2% PVP solution or a 0.5% Triton X-405 PBS solution, sample virus RNA is extracted in supernate, and the supernate is used for one-step RT-PCR amplification of target segments of the viruses, so that the melon viruses are detected. According to the RNA-RP RT-PCR method capable of fast detecting the melon viruses, only trace amounts of samples are needed for obtaining adequate amounts of RNA templates, the RNA templatesare used for detecting the six viruses of a Cucumber green mottle mosaic virus (CGMMV), a Cucumber mosaic virus (CMV), a Zucchini yellow mosaic virus (ZYMV), a Watermelon mosaic virus (WMV), a papayaring spot virus (PRSV) and a squash mosaic virus (SqMV) in watermelons, muskmelons, cucumbers, zucchini and a model plant nicotiana benthamiana in melon plants, the method is high in sensitivity and accurate in result, operation is simple and fast, and a novel detection method of the melon viruses is provided.

Description

technical field [0001] The invention belongs to the technical field of plant quarantine, in particular to an RNA-RP RT-PCR method for rapidly detecting melon viruses. Background technique [0002] Cucurbitaceae ( Cucurbitaceae ) plants include 118 genera and 825 species, and my country has 32 genera, 154 species and 35 varieties. my country's climatic conditions are suitable for the cultivation of cucurbit crops, and it is the world's largest planting country. Watermelons, melons, cucumbers and zucchini are the most common. Cucurbitaceae crop virus diseases are common, seriously affecting yield and quality. Viruses mainly include cucumber green mottle mosaic virus ( Cucumber green mottle mosaic virus , CGMMV), cucumber mosaic virus ( Cucumber mosaic virus , CMV), Zucchini Yellow Mosaic Virus ( Zucchini yellow mosaic virus , ZYMV), watermelon mosaic virus ( Watermelon mosaic virus , WMV), papaya ringspot virus ( Papaya ring spot virus , PRSV) and squash mosaic v...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2521/107
Inventor 刘莉铭古勤生
Owner ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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