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Living pseudomonas syringae pv.actinidiae detection method

A technology for canker bacteria and kiwifruit, which is applied in the field of detecting live bacteria of kiwifruit canker bacteria, can solve the problems of difficult operation and promotion, difficult detection of target bacteria, false positive detection, etc., so as to reduce the steps of cell lysis and DNA extraction, and save detection time. and cost, reducing false positives and false negatives

Inactive Publication Date: 2016-07-20
CHONGQING THREE GORGES UNIV
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Problems solved by technology

At present, PCR technology has been widely used in the detection of canker sores in kiwifruit, but the traditional PCR detection technology can not only amplify the DNA of live bacteria, but also the DNA of free states or dead bacteria, resulting in false positives in the detection
Moreover, the current detection method uses bacteria as the detection sample, and cannot directly use the kiwi fruit tree as the detection object. The target bacteria are not easy to be detected by taking the plant as the object, which is difficult, has many interference factors, and is not easy to operate and promote.

Method used

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  • Living pseudomonas syringae pv.actinidiae detection method

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Embodiment 1

[0024] Reagents: EMA (Invitrogen), Taq polymerase (TaKaRa), and conventional PCR reagents were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.

[0025] A method for detecting live bacteria of kiwifruit canker bacterium, which is operated according to the following steps:

[0026] (1) Clean the branches of the kiwi fruit to be tested and cut them into small pieces of 4 mm × 4 mm, wash them with sterile water for 3 times, then disinfect them with 1% sodium hypochlorite and 70% ethanol for 1 min, and rinse them with sterile water for 3 times;

[0027] (2) After crushing the sample under sterile conditions, add 0.8 mL of sterile distilled water to soak for 2 hours, take 0.4 mL of the upper soaking solution and inoculate it into 10 mL of LB liquid medium, and incubate on a shaking table at 25°C and 250 r / min for 8 hours;

[0028] (3) Take the culture solution after proliferation culture and add EMA at a final concentration of 3 μg / mL in the dark; treat the culture solutio...

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Abstract

The invention provides a living pseudomonas syringae pv.actinidiae detection method.The living pseudomonas syringae pv.actinidiae detection method uses detected kiwi fruit branches as detection samples and comprises the steps of dicing, cleaning and disinfection, proliferation culture, exposure and other steps, a specific primer, a reagent, process parameters and the like which are adopted are specifically disclosed, and the method can specifically, rapidly and accurately detect pseudomonas syringae pv.actinidiae in the branch samples and overcomes false positives and false negatives of a conventional PCR method.

Description

technical field [0001] The invention relates to a method for detecting live bacteria of kiwifruit canker bacterium. Background technique [0002] Kiwifruit canker is caused by Pseudomonas syringa epv. actinidiae (Psa), and is one of the most serious diseases in kiwifruit production. At present, PCR technology has been widely used in the detection of canker sores in kiwifruit, but the traditional PCR detection technology can not only amplify the DNA of live bacteria, but also the DNA of free state or dead bacteria, resulting in false positive detection. Moreover, the current detection method uses bacteria as the detection sample, and cannot directly use the kiwi fruit tree as the detection object. The target bacteria for detection with the plant as the object are not easy to be detected, are difficult, have many interference factors, and are not easy to operate and promote. [0003] Ethidiummonoazidebromide (EMA) is a dye that can bind to nucleic acid. When photolyzed, the n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/689C12Q1/686C12Q2600/158C12Q2563/173C12Q2565/125
Inventor 周大祥熊书
Owner CHONGQING THREE GORGES UNIV
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