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Real-time fluorescence PCR method for quantitatively detecting Pseudomonas syringae pv. Actinidiae

A technology of canker bacteria and real-time fluorescence is applied in the field of plant pathogen detection to achieve high sensitivity, improve detection efficiency and sensitivity, and prevent the expansion of the affected area

Inactive Publication Date: 2017-04-26
ZHEJIANG WANLI UNIV
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At present, the real-time fluorescence quantitative PCR method is used to detect the pathogenic bacteria in the country has no patent report yet

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  • Real-time fluorescence PCR method for quantitatively detecting Pseudomonas syringae pv. Actinidiae
  • Real-time fluorescence PCR method for quantitatively detecting Pseudomonas syringae pv. Actinidiae
  • Real-time fluorescence PCR method for quantitatively detecting Pseudomonas syringae pv. Actinidiae

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Embodiment

[0033] A real-time fluorescent PCR method for quantitative detection of kiwifruit canker bacterium, comprising the following steps:

[0034]Step a, extract the kiwifruit sample genomic DNA; electrophoresis detection of the band size and purity of the kiwifruit sample genomic DNA; in the step a, adopt the plant genome extraction kit to extract the kiwifruit sample genomic DNA of the suspected diseased kiwifruit branches or leaves, extract The purity of the genomic DNA band of the kiwifruit sample was detected by a micro-nucleic acid analyzer, and used for the fluorescent quantitative PCR amplification template in step b.

[0035] Step b, fluorescent quantitative detection of PSA pathogenic bacteria; perform fluorescent quantitative PCR amplification based on specific primers, and preliminarily determine whether PSA pathogenic bacteria are contained according to the amplification curve; the specific primers in the step b include upstream primer P3F: 5'-GGTTTCGGACACCGCAGGTTTCTACCG...

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Abstract

The invention discloses a real-time fluorescence PCR method for quantitatively detecting Pseudomonas syringae pv. Actinidiae. The method comprises the following steps: extracting an Actinidia chinensis sample genome DNA; electrophoretically detecting the band size and purity of the Actinidia chinensis sample genome DNA; carrying out fluorescent quantitative detection on a PSA pathogen; carrying out specific primer-based fluorescent quantitative amplification, and preliminarily determining whether the PSA pathogen is contained or not according to an amplification curve; purifying a PCR product with an obvious amplification peak, and sequencing the purified PCR product; and comparing a sequence obtained after the sequencing by Internet to further determine the PSA pathogen. The method allows a fluorescent quantitative PCR-based rapid detection method of the pathogen to be established, realizes rapid identification of the Pseudomonas syringae pv. Actinidiae infected sample, has the characteristics of simplicity in operation, high sensitivity, high specificity, and accurate and objective detection result, and is of great significance to improve the detection efficiency and the detection sensitivity of the Pseudomonas syringae pv. Actinidiae infected sample.

Description

technical field [0001] The invention belongs to the technical field of plant pathogen detection, and in particular relates to a real-time fluorescent PCR method for quantitatively detecting kiwifruit canker bacterium. Background technique [0002] Kiwifruit (Actinidia chinensis) is a fruit with high economic value, which is widely planted at home and abroad. However, in recent years, with the continuous increase of kiwifruit cultivation area, bacterial canker has spread rapidly. Since 2005, it has caused serious harm in the world's major kiwifruit producing areas and has become the most devastating disease in the kiwifruit cultivation process, seriously threatening the world. The production of kiwi is safe. Kiwifruit canker is a devastating bacterial disease that was first discovered in Japan and the United States in the early 1980s and reported in detail. Subsequently, the disease appeared in South Korea, Iran, Italy and other countries one after another. Beginning in 20...

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q1/6869C12Q1/689C12Q2561/113C12Q2563/107C12Q2545/114C12Q2531/113
Inventor 俞超谢锦添吴月燕马立孟
Owner ZHEJIANG WANLI UNIV
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