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One-step RT-PCR (Reverse Transcription Polymerase Chain Reaction) detection reagent kit for Cucumber green mottle mosaic virus and detection method thereof

An RT-PCR and detection kit technology, applied in the field of one-step RT-PCR detection kits for cucumber green mottle mosaic virus, can solve the problems of low reliability, long detection period, complicated processing, etc. The effect of reducing detection cost and simple production method

Inactive Publication Date: 2010-09-29
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CGMMV can be identified by inoculating indicator plants such as quinoa, datura, petunia, etc., but the symptoms are very similar to other viruses of the same genus, so the reliability is not high, and the detection cycle is long
The detection of CGMMV by electron microscopy not only requires special equipment, but also the pretreatment of materials is relatively complicated.
Serological detection has the advantages of simple equipment, easy operation, low cost and sensitive response, but it is still difficult to detect by serological detection when the sample amount is very small or the virus content in the tissue is very low
Compared with serological detection methods, RT-PCR molecular detection methods based on nucleic acid levels have significant advantages such as higher sensitivity, stronger specificity, and more reliable results, but so far, there is no one-step RT-PCR detection reagent for CGMMV The report of the box and its detection method

Method used

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  • One-step RT-PCR (Reverse Transcription Polymerase Chain Reaction) detection reagent kit for Cucumber green mottle mosaic virus and detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0030] Cucumber green mottle mosaic virus one-step RT-PCR detection kit, is characterized in that, said kit comprises:

[0031] 1)A # Tube containing the upstream primer of cucumber green mottle mosaic virus, the concentration is 10 μmol / L, and the primer sequence is 5'-TGCTTCTTATGTTCCCGTCA-3';

[0032] 2)B # Tube containing the downstream primer of cucumber green mottle mosaic virus, the concentration is 10 μmol / L, and the primer sequence is 5'-GCCCATAGAAACTTCAACGTC-3';

[0033] 3)C # tube, filled with PCR Buffer, the concentration is 10×;

[0034] 4)D # tube, containing Mgcl 2 , the concentration is 25mmol / L;

[0035] 5)E # tube, filled with dNTPs, the concentration is 10mmol / L;

[0036] 6)F # tube, filled with RNase inhibitor, the concentration is 40U / μL;

[0037] 7) G # Tube, filled with reverse transcriptase, the concentration is 5U / μL;

[0038] 8)H # tube, containing DNA polymerase at a concentration of 5 U / μL;

[0039] 9)I # Tube containing positive contr...

Embodiment 2

[0044] The detection method of the cucumber green mottle mosaic virus one-step RT-PCR detection kit comprises the steps:

[0045] 1) According to the number of samples to be tested, add A to each PCR tube # Solution 2μL, B # Solution 2μL, C # Solution 5μL, D # Solution 10μL, E # Solution 5μL, F # Solution 1μL, G # Solution 1μL, H # Solution 1μL and K # 21 μL of solution, mix well and centrifuge for later use;

[0046] 2) RT-PCR reaction: Take 2 μL each of the sample to be tested, the negative control and the positive control RNA template, and add them to each PCR tube prepared in step (1), mix well, and perform the following RT-PCR reaction after centrifugation: first cDNA was synthesized at 42°C for 30 minutes, followed by pre-denaturation at 94°C for 5 minutes, and then entered into the following cycle: denaturation at 94°C for 30 s, annealing at 54°C for 45 s, and extension at 72°C for 1 min, a total of 35 cycles; after the last cycle, extend at 72°C for 10 min;

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Abstract

The invention relates to a one-step RT-PCR (Reverse Transcription Polymerase Chain Reaction) detection reagent kit for Cucumber green mottle mosaic virus and a detection method thereof. The reagent kit comprises the following various agents for a one-step RT-PRC reaction: an upstream primer of Cucumber green mottle mosaic virus, a downstream primer of Cucumber green mottle mosaic virus, a PCR Buffer, Mgc12, dNTPs, an RNA enzyme inhibitor, a reverse transcriptase, a DNA polymerase, positive control containing Cucumber green mottle mosaic virus and negative control containing no Cucumber green mottle mosaic virus and RNase-free ddH2O. The invention has the advantages of simple preparation method and convenient large-scale industrialized production, and the detection method has the advantages of simple and convenient operation, high sensitivity, strong specificity and good accuracy. The whole detection process is accomplished in a PCR tube and suitable for rapid detection of a batch of samples, thereby simplifying the operation procedure, reducing the detection time, avoiding the cross contamination, and improving the detection efficiency.

Description

technical field [0001] The invention relates to a one-step RT-PCR detection kit for cucumber green mottle mosaic virus and a detection method thereof, which belong to the field of biotechnology and are specially used for rapid detection of cucumber green mottle mosaic virus by port inspection and quarantine and agricultural plant inspection departments. Background technique [0002] Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus (Cucumber green mottle mosaicvirus, Tobamovirus), and the virus genome is a positive single-stranded RNA with a total length of about 6.5kb. CGMMV takes watermelon (Citrullus lanatus), cucumber (Cucumis sativus), muskmelon (Cucumis melo), gourd (Iagenaria siceraria), pumpkin (Cucurbita moschata) and other cucurbit crops as its main hosts. CGMMV is currently mainly distributed in Europe, Asia and South America, including the United Kingdom, Germany, the Netherlands, Denmark, Russia (former Soviet Union), Greece, Austri...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/94
Inventor 翁瑞泉沈建国王念武翁泽斌陈劲松林双庆
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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