海产品中副溶血性弧菌免疫磁分离和环介导等温扩增快速检测方法的建立
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张蕾 女 硕士生 研究方向为食品安全与控制 E-mail:zlei0605@yahoo.cn

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国家质检总局公益性行业科研专项(201110034),国家质检总局科技计划项目(2009IK184)


A noval method of nano-immunomagnetic separation and loop-mediated isothermal amplification for detecting Vibrio parahaemolyticus from seafoods
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    摘要:

    采用纳米免疫磁珠分离副溶血性弧菌,建立副溶血性弧菌环介导等温扩增检测方法。方法 采用副溶血性弧菌单克隆抗体,制备纳米免疫磁珠,特异性吸附副溶血性弧菌,结合环介导等温扩增技术,建立副溶血性弧菌快速检测方法。结果 副溶血性弧菌纳米免疫磁珠在菌体浓度为103cfu/ml水平时,对副溶血性弧菌的捕获率达到74%。免疫磁分离结合环介导等温扩增技术,在纯培养、无需增菌情况下,检测灵敏度达到140cfu/ml增菌液;通过对134株副溶血性弧菌和74株非目标菌的测试,环介导等温扩增技术具有良好的特异性;食品基质添加试验中,在增菌时间缩短至8h的条件下,其检测限为2cfu/25g样品。结论 副溶血性弧菌免疫纳米磁珠结合环介导等温扩增技术,有效缩短了增菌时间,适用于副溶血性弧菌的快速检测。

    Abstract:

    Anoval method of nano-immunomagnetic separation (Nano-IMS) plus loop-mediated isothermal amplification (LAMP) was established for the rapid detection of Vibrio parahemolyticus.MethodsThe Nano-Immunomagnetic Beads (Nano-IMB) were created by using the monoclonal antibody of V. parahemolyticus and named Nano-IMB-Vp, which was highly specific to V. parahemolyticus. The method of Nano-IMS-LAMP was established for the rapid detection of V. parahemolyticus from seefoods. ResultsThe capture ratio of Nano-IMB-Vp was reached 74% at the level of 103cfu/ml. In pure culture, the sensitivity of Nano-IMS-LAMP was reached 140cfu/ml culture medium. The specific of this LAMP method was tested by using 134targets and 74non-targets bacteria. The results showed that the LAMP method was highly specific to V. parahemolyticus. Nocross-reaction was founded. The detection limit of Nano-IMS-LAMP reached 2cfu/25g in artficial samples when the period of time for enrichment was shorted to 8h.ConclusionThe method of Nano-IMS-LAMP could shorten the period of time for enrichment effectively, which can apply to the rapid detection of V. parahemolyticus.

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张蕾,张海予,曾静,程晋霞,魏海燕,张西萌,马丹.海产品中副溶血性弧菌免疫磁分离和环介导等温扩增快速检测方法的建立[J].中国食品卫生杂志,2013,25(5):401-405.

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  • 收稿日期:2013-06-17
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  • 在线发布日期: 2013-10-25
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