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Primer and probe for detecting cucumber green mottle mosaic virus

A green mottled flower and leaf virus technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of loss of edible and commercial value, discoloration of watermelon fruit pulp, economic loss, etc. To achieve the effect of high accuracy, high sensitivity and high sensitivity

Inactive Publication Date: 2008-05-28
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the reported CGMMV isolates are watermelon strains (CGMMV-W). After CGMMV-W infects watermelon, it can cause typical mottled and mosaic symptoms, and it can also cause discoloration and rot inside the watermelon fruit during the ripening stage. Losing edible and commercial value, causing significant economic losses, the virus has been included in my country's newly revised list of harmful organisms prohibited from entering the country

Method used

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  • Primer and probe for detecting cucumber green mottle mosaic virus
  • Primer and probe for detecting cucumber green mottle mosaic virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Design and synthesis of embodiment 1 primers and probes

[0018] According to the public sequences of the two virulence factors MRP (GenBank No.X64450) and EF (GenBank No.A24023) coding genes of Streptococcus suis type 2, primers and probes were designed using the probe design software Express 2.0. The primer sequences are:

[0019] CGMMV-RP (Forward): 5'-GCATAGTGCTTTCCCGTTCAC-3'

[0020] CGMMV-FP (reverse): 5'-TGCAGAATTACTGCCCATAGAAAC-3'

[0021] The sequence of the probe is: 5'-CGGTTTGCTCATTGGTTTGCGGA-3'

[0022] The 5' end of the probe was labeled with the reporter fluorescent dye FAM, and the 3' end was labeled with the quencher fluorescent dye TAMRA.

Embodiment 2

[0023] The extraction of embodiment 2 total RNA

[0024] 1) Take 0.1g of diseased leaves, cut into small pieces, grind into powder with liquid nitrogen, transfer to a sterilized 1.5ml centrifuge tube, then add 1ml of Trizol reagent, shake vigorously;

[0025] 2) Centrifuge at 12000g for 10min at 4°C to remove insoluble components, and transfer the supernatant to a new 1.5ml centrifuge tube;

[0026] 3) Keep at room temperature for 5 minutes, add 0.2ml of chloroform, shake vigorously for 15s, then keep at room temperature for 2-15 minutes, then centrifuge at 12000g for 15 minutes at 4°C;

[0027] 4) Transfer the upper aqueous phase to a new 1.5ml centrifuge tube, add 0.5ml isopropanol, invert and mix, and keep at room temperature for 15 minutes;

[0028] 5) Centrifuge at 12000g for 10min at 4°C, and the RNA will form a precipitate on the side wall and bottom of the tube;

[0029] 6) Pour off the supernatant, add 75% ethanol to wash the precipitate, then centrifuge at 7500g fo...

Embodiment 3

[0031] The establishment of embodiment 3 Real-time RT-PCR amplification method

[0032] 1. Real-time fluorescent RT-PCR reaction system

[0033] Real-time fluorescent RT-PCR reaction was performed using total RNA as a template, that is, 20.25 μL DEPC-treated water, 25 μL 2×Master Mix withoutUNG, 1.25 μL 40×MultiScribe and RNase Inhibitor Mix, 1 μL primer CGMMV-FP ( 20 μmol / L), 1 μL primer CGMMV-RP (20 μmol / L), 0.5 μL probe CGMMV-FAM (20 μmol / L), 1.6ng total RNA.

[0034] 2. Real-time fluorescent RT-PCR reaction conditions

[0035] After putting the sample tube into the ABI 7700 fluorescent PCR instrument of ABI Company, set the following conditions for reaction: 42°C, 30min; 95°C, 5min; 95°C, 15s; 60°C, 1min, a total of 40 cycles. Collect data after each cycle. After the reaction, judge the result according to the amplification curve.

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Abstract

The invention provides a primer and a probe for detecting cucumber green mottle mosaic virus. The nucleotide sequence of the primer is shown as the sequence table SEQ ID NO.1&2, and the nucleotide sequence of the probe is shown as a sequence table SEQ ID NO.3. The invention further provides a method for detecting cucumber green mottle mosaic virus, the method takes the general RNA of samples as templates, conducts real-time fluorescence RT-PCR amplification by employing the primer and probe, each cycle finishes data collection, and the invention determines the result according to the amplification curve after reaction. The invention has primer with perfect specificity, rapid and simple detecting method, high accuracy and sensibility, which ensures import and export security.

Description

technical field [0001] The invention relates to biological detection technology, in particular to primers and probes for detection of cucumber green mottled mosaic virus. Background technique [0002] Cucumber green mottle mosaic virus (CGMMV) is one of the important viruses of the genus Tobamovirus in Cucurbitaceae, which poses a serious threat to the production of Cucurbitaceae crops. Cause stains, blisters and deformation on cucumber leaves, dwarf the plants, delay the results, most of the fruits yellow or turn white and produce black-green blister-like necrotic spots, the yield is 15%; in the Georgia area of ​​the former Soviet Union, The incidence rate is as high as 80% to 100%, which can delay the growth and development of cucumber plants, and even cause infertility. The watermelon plants infected in the early stage grow slowly, appear mosaic, the inside of the fruit is seriously discolored or cause rot, the pulp is fibrous, and the yield is reduced by 30%. In 1998, t...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 赵文军陈红运白净朱水芳李明福
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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