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PCR primer for banana bunchy top virus detection method and detection kit thereof

A technology of banana bunchy top virus and detection primers is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. High detection sensitivity, simplified operation process, and the effect of reducing pollution

Inactive Publication Date: 2009-07-22
INSPECTION AND QUARANTINE TECHNOLOGY CENTER ZHONGSHAN ENTRY EXIT INSPECTION AND QUARANTINE +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] BBTV is a DNA virus. At present, some people use PCR to detect banana bunchy top virus, but the related kits generally add PCR reagents in multiple steps, which is not only very easy to cross-contaminate, but also time-consuming and laborious, and the detection sensitivity is not high.

Method used

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  • PCR primer for banana bunchy top 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% ethanol;

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

[0041] (10) The precipitate was dissolved in 50 uLTE (pH 8.0) an...

Embodiment 2

[0068] Embodiment two: the preparation of banana bunchy top virus PCR kit

[0069] The composition of the kit is as follows:

[0070] DNA Polymerase Mixture (DNA Polymerase Mixture) 25μl×24 sticks

[0071] BBTV upstream primer 30μl

[0072] BBTV downstream primer 30μl

[0073] Banana bunchy top virus positive control 50μl (2.5μg)

[0074] 25 μl of DNA polymerase mixture × 24 tubes, for the 50 μl PCR reaction system, use 1 tube each time, that is, 25 μl. The primer concentration in the kit is 50 μM, and the primer concentration is diluted to 1 μM when used.

[0075] The kit of the present invention comprises the DNA polymerase mixture (DNA Polymerase Mixture) (purchased from Takara Company) specially used for the PCR reaction of banana bunchy top virus (BBTV), which has been subpackaged to 0.2ml Micro PCR tube (25 μl / branch) In use, as long as the nucleic acid of the sample to be tested and the BSV primer in this kit are added, the PCR reaction can be carried out, which gr...

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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 BBTV-P1:5'-ATGTGGTATGCTGGATGTTC-3' and a downstream primer BBTV-P2:5'-GTTCATATTTCCCGCTTTGA-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 bunchy top 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 prevention...

Claims

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Application Information

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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