多重LAMP—熔解曲线法检测食品中两种食源性致病菌
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(浙江省质量检测科学研究院,浙江 杭州 310018)

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姜侃(1978—),男,浙江省质量检测科学研究院高级工程师。E-mail:jjj_john@163.com

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浙江省科技厅优先主题重点发展项目(编号:2009C13013);浙江省质监系统科研项目(编号:20120101)


Development of multiplex LAMP combined with melting curves analysis for detection of two kinds of food borne pathogens
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(Zhejiang Insistitute of Quality Inspection Science, Hangzhou, Zhejiang 310018, China)

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    摘要:

    针对沙门氏菌的侵袭蛋白基因(invA)和金黄色葡萄球菌的耐热核酸酶基因(nuc)设计LAMP引物,优化反应条件,并分别对反应产物进行熔解曲线分析,判断扩增结果,从而建立食品中沙门氏菌和金黄色葡萄球菌多重LAMP—熔解曲线检测方法。对9株目标菌和23株非目标菌的检测未出现假阳性和假阴性结果,并可通过熔解曲线的熔解温度分析确定所含目标菌;对产物的测序分析表明所得序列与目的基因序列吻合;对目标菌的检测灵敏度均可达到10 fg/μL;对232份样品的检测结果显示,其中2份生猪肉中检出金黄色葡萄球菌。该方法具有良好的检测特异性,并可为食源性致病菌的快速检测提供一种重要技术手段。

    Abstract:

    A multiplex LAMP-melting curve method for simultaneous detection of Salmonella spp. and Staphylococcus aureus in food was developed. The primers were designed according to the sequences of invA gene of Salmonella spp. and nuc gene of Staphylococcus aureus, and the reaction system was optimized. The amplification products were analyzed by melting curves, electrophoresis, precipitation observation and fluorescence observation as well. No false positive and false negative results emerged during tests of 9 target and 23 non-target bacteria strains, and the pathogens could be determined by melting temperature on melting curves. DNA sequencing of LAMP products showed the sequences as deduced, confirming the correct LAMP amplification. The detection limits for each pathogen were both 10 fg/μL. The tests result of 232 samples showed that 2 pieces of raw pork were contaminated by Staphylococcus aureus. This method showed high specificity, and could be used to detect foodborne pathogens rapidly.

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引用本文

姜侃,张慧,汪新,等.多重LAMP—熔解曲线法检测食品中两种食源性致病菌[J].食品与机械,2015,31(2):87-92.
JIANGKan, ZHANGHui, WANGXin, et al. Development of multiplex LAMP combined with melting curves analysis for detection of two kinds of food borne pathogens[J]. Food & Machinery,2015,31(2):87-92.

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  • 收稿日期:2014-12-15
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  • 在线发布日期: 2023-03-17
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