应用SYBR GreenⅠ溶解曲线检测食源性单增李斯特菌
Detection of Listeria monocytogenes in foods by SYBR GreenⅠmelting curve
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摘要: 基于单增李斯特菌hlyA基因和李斯特菌属的16sRNA基因分别合成两对引物,确立SYBR GreenⅠ荧光PCR检测单增李斯特菌两个特异性基因的最佳反应体系及条件,反应液经凝胶电泳验证,验证结果显示,扩增出的两条不同大小、不同Tm值的目的核酸片段,分别对应两条荧光PCR溶解曲线上的两个独立波峰。基于单增李斯特菌hlyA基因建立标准曲线,相关系数为0.996,最低检出限约为89CFU/mL。为检验方法的可行性,对鲜活白蚬子样品中分离出的疑似菌株进行实际检测,所得阳性结果经国家标准方法（GB4789.30-2010）验证,验证结果表明,SYBR GreenⅠ荧光PCR实际检测阳性结果与GB4789.30-2010检测结果一致。Abstract: Two pairs of specific primers were composited based on the hlyA gene of L.monocytogenes and the 16sRNA gene of the genus Listeria.Optimal reaction system and condition of the SYBR GreenⅠfluorescent PCR were established, respectively, which could detect two specific genes.The reaction solution was verified by the method of gel electrophoresis.The result showed that two target fragments possessed different sizes and Tm valves which were amplfied corresponded two separate peaks.The standard curve based on the L.monocytogenes hlyA gene was established and the correlation coefficient was 0.996.The minimal detection limit was 89CFU/mL.Moreover, suspicious strain isolated from white clams son was tested by using this method so as to study the feasibility.The positive result was evaluated with the National Standard Methods (GB 4789.30-2010) .The verified result indicated that the positive result of SYBR GreenⅠfluorescent PCR matched with the GB 4789.30-2010 exactly.
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