王虹玲, 朱水荣, 梅玲玲. 副溶血弧菌的gyrB基因环介导的恒温扩增技术检测方法的研究[J]. 疾病监测, 2009, 24(8): 621-624. DOI: 10.3784/j.issn.1003-9961-2009.08.024
引用本文: 王虹玲, 朱水荣, 梅玲玲. 副溶血弧菌的gyrB基因环介导的恒温扩增技术检测方法的研究[J]. 疾病监测, 2009, 24(8): 621-624. DOI: 10.3784/j.issn.1003-9961-2009.08.024
WANG Hong-ling*, ZHU Shui-rong, MEI Ling-ling. Study on loop-mediated isothermal amplification (LAMP) technique for detection of <I>Vibrio parahaemolyticus</I> based on the<I>gyrB</I> gene[J]. Disease Surveillance, 2009, 24(8): 621-624. DOI: 10.3784/j.issn.1003-9961-2009.08.024
Citation: WANG Hong-ling*, ZHU Shui-rong, MEI Ling-ling. Study on loop-mediated isothermal amplification (LAMP) technique for detection of <I>Vibrio parahaemolyticus</I> based on the<I>gyrB</I> gene[J]. Disease Surveillance, 2009, 24(8): 621-624. DOI: 10.3784/j.issn.1003-9961-2009.08.024

副溶血弧菌的gyrB基因环介导的恒温扩增技术检测方法的研究

Study on loop-mediated isothermal amplification (LAMP) technique for detection of <I>Vibrio parahaemolyticus</I> based on the<I>gyrB</I> gene

  • 摘要: 目的建立一种适合基层实验室应用和开展的快速检测副溶血弧菌的DNA环介导的恒温扩增法(LAMP)。方法针对副溶血弧菌的IgyrB/I 基因序列设计了4条引物(2条内引物、2条外引物),并对扩增反应条件进行了优化。结果整个检测过程仅需1.5 h,可通过肉眼目测或电泳检 测判断结果;对3株种系背景明确的副溶血弧菌不同实验对照株、23株副溶血弧菌地方分离株和32株其他肠道菌进行了检测,具有很高的特异性 ;该方法的最低检测限为24 cfu/ml,具有良好的敏感性;应用于61份贝类海产品的现场检测,阳性率为100%,与实时荧光定量PCR方法的检测 结果相符,阳性率高于传统的培养鉴定方法。结论本方法具有快速、灵敏、特异、简便、经济等特点,适合基层实验室、应急检测或现场监测 等使用,具有较高的推广价值。

     

    Abstract: ObjectiveTo establish a method of loop-mediated isothermal amplification to detect IVibrio parahaemolyticus/I for the laboratory at grassroots unit. MethodsFour primers (two inner primers, two outer primers) were designed specifically to recognize the IgyrB/I gene of IVibrio parahaemolyticus/I. ResultsIt only takes 1.5 h to conduct the detection with optimized condition and the result can be judged by naked eyes or electrophoresis. Three strains of different species IV. Parahaemolyticus/I, 23 local strains of IV. parahaemolyticus/I and 32 strains of other intestinal bacteria were detected by the method with high specificity. The method was sensitive with the lowest detectable limit (LDL) of 24 cfu/ml. In the field detection of 60 shellfishes, the positive rate was 100%, which was higher than that of traditional method and was consistent with the result of RT-PCR. ConclusionThe method is suitable for the laboratory at grassroots unit, emergency detection and field detection, which is rapid, sensitive, specific, simple, economical, which and is worth to spread.

     

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