PCR primer for heart rot mosaic virus detection method and detection kit thereof

A technology for detection of primers for banana mosaic and leaf core rot, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve the problems of low detection sensitivity, time-consuming and labor-intensive, and easy cross-contamination, etc. Achieve high detection sensitivity, simple and convenient operation, and prevent cross-contamination

Inactive Publication Date: 2009-07-22
INSPECTION AND QUARANTINE TECHNOLOGY CENTER ZHONGSHAN ENTRY EXIT INSPECTION AND QUARANTINE +2
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Problems solved by technology

[0005] At present, some people use PCR to detect cucumber mosaic virus, but the two-step method used, that is, the reverse transcription RT step and the PCR amplification step are carried out separately, which is not only very easy to cross-contamination, but also time-consuming and laborious, and the detection sensitivity is not high.

Method used

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  • PCR primer for heart rot mosaic virus detection method and detection kit thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. Extract banana total nucleic acid (DNA+RNA)

[0032] (1) Take 0.1-0.2g of tender leaves to be tested, place them in a 1.5mL centrifuge tube, and freeze them with liquid nitrogen;

[0033] (2) Vigorously oscillate by an oscillator, and grind the blade with a glass rod;

[0034] (3) Add 0.6mL CTAB extraction buffer homogenate + 2% 2-ME, shake and mix;

[0035] (4) Incubate at 65°C for 30 minutes; then add 0.5 mL of chloroform and mix for 5 minutes;

[0036] (5) Centrifuge at 12000rpm for 5min, and transfer the upper aqueous phase to a new centrifuge tube;

[0037] (6) Add 1× volume of isopropanol, mix gently, and place at -20°C for 30 minutes;

[0038] (7) centrifuge at 12000rpm for 10min, discard the supernatant;

[0039] (8) Wash the precipitate with 70% ethanol, centrifuge at 12000rpm for 2min; then wash once with 100% ethyl alcohol;

[0040] (9) Discard the supernatant, and vacuum-dry for 5 minutes;

[0041] (10) The precipitate was dissolved in 50 uLTE (pH 8...

Embodiment 2

[0069] Embodiment two: the preparation of banana bunchy top virus PCR kit

[0070] The composition of the kit is as follows:

[0071] 1. Enzyme Mix 100μl

[0072] 2. Buffer 625μl×2

[0073] 3. CMV upstream primer (Primer-1) 50 μl (concentration is 1 μm)

[0074] 4. CMV downstream primer (Primer-2) 50μl (concentration is 1μm)

[0075] 5. Banana mosaic heart rot virus positive control 50μl (50μg)

[0076] 6. RNase Free deionized water (RNase Free dH2O) 1000μl

[0077] The above is the amount of the RT-PCR reaction system for 50 times, each time 50 μl.

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Abstract

The invention provides a PCR detecting primer and a PCR detecting kit of cucumber mosaic virus, wherein, an upstream primer CMV-P1:5'-CACCCAACCTTTGTGGGTAG-3' and a downstream primer CMV-P2:5'-CAACACTGCCAACTCAGCTC-3' are adopted. The PCR detecting kit of the invention can greatly lower cross-contamination probability in detection, remarkably raises detection efficiency, has ultrahigh detection sensitivity as well as simple and convenient operation, and prevents cross-contamination.

Description

technical field [0001] The invention relates to a primer for detecting banana mosaic heart rot virus and a detection kit thereof. Background technique [0002] Banana is the pillar industry of Zhongshan's fruit industry and an important export agricultural product. It is exported to Japan and has a good reputation in the Japanese market. The annual export value is about 100,000 US dollars. At present, banana virus disease has always been one of the main limiting factors for banana production and the export of banana tissue cultured seedlings in my country. In recent years, banana virus disease has also had a serious impact on the yield and quality of bananas in Zhongshan City, and has become a major factor in the development of banana industry in Zhongshan City. one of the main limiting factors. In addition, once bananas are infected by viruses, it is difficult to control them with chemicals. To deal with banana virus diseases, the most effective measure is to focus on prev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 陈定虎王龙贵杨雷亮管维
Owner INSPECTION AND QUARANTINE TECHNOLOGY CENTER ZHONGSHAN ENTRY EXIT INSPECTION AND QUARANTINE
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