环介导等温扩增方法在食品中沙门菌检测的应用和评价
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张蕾 女 硕士 研究方向为食品安全与控制 E-mail:zlei0605@yahoo.cn通讯作者:┣┣(中)通信作者┫┫曾静 女 研究员 研究方向为食品微生物学 E-mail:zengj@bjciq.gov.cn

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国家质检总局公益性行业科研专项(201110034)


Application and evaluation of loop-mediated isothermal amplification method for detceting of Salmonella spp. in food
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    摘要:

    将环介导等温扩增检测方法应用于食品中沙门菌的检验,并在检测方法特异性、灵敏度等方面与实时荧光PCR和传统检测方法进行比较。方法 针对沙门菌属高度保守的fimY基因设计环介导等温扩增检测引物并优化反应体系,在特异性、灵敏度和实际样品检测等方面与实时荧光PCR及传统检测方法比对。结果 本研究建立的LAMP方法检测沙门菌93株和非目标菌31株,具有良好的特异性。在纯培养、无需增菌情况下,其检测灵敏度为6.4×102cfu/ml,与实时荧光PCR方法相当。食品基质添加试验中,环介导等温扩增方法检测低限为2cfu/25g样品;对45份实际食品样品检测结果表明,该方法实际样品检出率为11.1%,与实时荧光PCR及传统方法检测结果一致。结论 本研究建立的沙门菌环介导等温扩增检测方法具有良好的特异性,检测灵敏度与实时荧光PCR相当,适用于沙门菌的快速筛选。

    Abstract:

    The loop-mediated isothermal amplification (LAMP) detection method was applied to detect Salmonella spp.in food. The specificity and sensitivity of this method were compared with real-time PCR and conventional detection method.MethodsThe fimY gene of Salmonella spp. was used to design LAMP primers, and then optimized LAMP reaction system. LAMP method was compared with real-time PCR and conventional detection methods in some aspects, such as specificity, sensitivity and practical food samples detection. ResultsThe specificity of LAMP method was tested by using 93targets and 31non-targets bacteria. The results showed that the LAMP method was highlyspecific to Salmonella spp.. No cross-reaction was founded. In pure culture, the sensitivity of LAMP was 6.4×102cfu/ml, which was consistent with real-time PCR method. The detection limit of LAMP reached 2cfu/25g in base-material addition test. The detection of 45practical food samples showed the detection rate of LAMP was 11.1%, which was as same as real-time PCR and traditional methods.ConclusionThe LAMP detection method of Salmonella spp. established in this study has good specificity and sensitivity, which can apply to the rapid detection of Salmonella spp..

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张蕾,张海予,魏海燕,张西萌,程晋霞,曾静.环介导等温扩增方法在食品中沙门菌检测的应用和评价[J].中国食品卫生杂志,2013,25(6):520-524.

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  • 收稿日期:2013-06-25
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  • 在线发布日期: 2013-12-31
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