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首页> 外文期刊>Journal of Pure & Applied Microbiology >Establishment of Real-time Quantitative PCR Method for Vibrio parahaemolyticus in Sediment of the Stichopus japonicus Culture Pond
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Establishment of Real-time Quantitative PCR Method for Vibrio parahaemolyticus in Sediment of the Stichopus japonicus Culture Pond

机译：刺猬养殖池沉淀物中副溶血性弧菌实时定量PCR方法的建立

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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.
机译：通过使用针对toxR基因的特异性引物，开发了一种快速实时定量PCR方法，用于对刺参培养池沉淀物中的副溶血性弧菌进行分析。将已知数量的副溶血性弧菌细胞掺入无菌沉淀物中，作为模拟沉淀物样品。尝试了三种改良的沉淀物DNA提取方法，证明了改良的溶菌酶-SDS温和裂解法可用于沉淀物DNA提取。通过使用从模拟沉淀物中提取的连续稀释总DNA和从纯培养质粒中提取的连续DNA绘制标准曲线。此外，还很好地证明了该检测方法的灵敏度，特异性和可重复性。发现沉淀物中副溶血性弧菌的定量极限为10（2）cfu g（-1），并且由于所有变异系数（CV）值介于0.31％和0.92％之间，因此具有较高的可重复性，较少超过3％。沉积物中副溶血性弧菌实时PCR定量方法的发展对于培养日本刺猬的疾病预防具有重要意义。

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