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Establishment of Real-time Quantitative PCR Method for Vibrio parahaemolyticus in Sediment of the Stichopus japonicus Culture Pond

文献类型: 外文期刊

作者: Li Qiu-fen 1 ; Jiang Wei-wei 1 ; Liu Huai-de 1 ; Li Xiao-long 1 ;

作者机构: 1.Chinese Acad Fishery Sci, Yellow Sea Fisheries Res Inst, Qingdao 266071, Peoples R China

2.Ocean Univ China, Coll Marine Life Sci, Qingdao 266100, Peoples R China

关键词: toxR;Vibrio parahaemolyticus;Real-time Quantitative PCR;Stichopus japonicus culture pond;Sediment DNA extraction method

期刊名称:JOURNAL OF PURE AND APPLIED MICROBIOLOGY ( 影响因子:0.073; )

ISSN:

年卷期:

页码:

收录情况: SCI

摘要: A rapid real-time quantitative PCR method was developed for V. parahaemolyticus in sediment of the Stichopus japonicas culture pond by using specific primers targeted toxR gene. The known number V. parahaemolyticus cells were spiked into sterile sediment served as the simulative sediment samples. Three modified sediment DNA extraction methods were tried, it was proved that modified lysozyme-SDS gentle lyse method is feasible for the sediment DNA extraction. The standard curves was made by using serial diluted total DNA extracted from the simulated sediment and which from plasmid of pure culture. Besides, sensitivity, specificity and repeatability of the detection method were demonstrated well. The quantification limit was found to be 10(2) cfu g(-1) for V. parahaemolyticus in sediment, and have a high repeatability as a result of all the coefficient of variation (CV) values between 0.31% and 0.92%, less than 3%. Development of the real-time PCR quantification method for V. parahaemolyticus in the sediment was important for disease prevention the cultured Stichopus japonicas.

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