Ramírez Mérida Luís Guillermo, Morón de Salim Alba, Alfieri Graterol Ana Yudith, Gamboa Orlando
Departamento de Biología, Facultad de Ciencias y Tecnologia, Salud Universidad de Carabobo Sede Valencia, Venezuela.
Arch Latinoam Nutr. 2010 Sep;60(3):254-60.
The polymerase chain reaction, known as PCR, is a method to replicate thousands of times within a few hours and in vitro, small amounts of DNA. The application of rapid and sensitive methods to detect Listeria monocytogenes in cheese samples, allow a better microbiological control of the production process. PCR was applied to 30 samples of of white cheese, from Valencia, Carabobo State. It was detected PCR specificity and sensitivity by using the control strain Listeria monocytogenes 446. DNA extraction according to the methodology described by Torres et al., Molecular weight marker 100 base pairs. Were used: four primers hlyA gene of listeriolysin O; L1/U1 primers for 938 bp band and LF/LR 750 bp band hlyA gene. Epilnfo Statistical V6 to match observations in gels, by Kappa coefficient (K).
8 out of 30 cheese samples analyzed showed presumptive growth of Listeria spp in PALCAM Agar. Two of the samples not belonged to the genus Listeria, in the 6 remaining sample confirmation tests showed that: 2 were L. monocytogenes, 3 L. ivanovii and 1 L. seeligeri. In PCR 2 samples were positive for L. monocytogenes by amplify the 938 bp band for Listeria and 750 bp band for the species monocytogenes. We concluded that PCR was highly specific and sensitive to L. monocytogenes, taking advantage of PALCAM agar to detect the presence of the pathogen specifies a relatively short time.
聚合酶链反应，即PCR，是一种能在数小时内于体外将少量DNA复制数千倍的方法。应用快速灵敏的方法检测奶酪样品中的单核细胞增生李斯特菌，有助于对生产过程进行更好的微生物控制。对来自卡拉沃沃州巴伦西亚的30份白奶酪样品进行了PCR检测。通过使用单核细胞增生李斯特菌446对照菌株检测PCR的特异性和灵敏度。根据托雷斯等人描述的方法进行DNA提取，分子量标记为100碱基对。使用了：四种用于溶血素O的hlyA基因的引物；用于938 bp条带的L1/U1引物和用于hlyA基因750 bp条带的LF/LR引物。使用Epilnfo统计软件V6通过卡帕系数（K）匹配凝胶中的观察结果。
在分析的30份奶酪样品中，有8份在PALCAM琼脂上显示出李斯特菌属的推定生长。其中两份样品不属于李斯特菌属，在其余6份样品的确认试验中表明：2份为单核细胞增生李斯特菌，3份为伊氏李斯特菌，1份为斯氏李斯特菌。在PCR检测中，有2份样品通过扩增李斯特菌的938 bp条带和单核细胞增生李斯特菌的750 bp条带对单核细胞增生李斯特菌呈阳性。我们得出结论，PCR对单核细胞增生李斯特菌具有高度特异性和敏感性，利用PALCAM琼脂在相对较短的时间内检测病原体的存在。
