Furrer B, Candrian U, Hoefelein C, Luethy J
Institute of Biochemistry, University of Berne, Switzerland.
J Appl Bacteriol. 1991 May;70(5):372-9. doi: 10.1111/j.1365-2672.1991.tb02951.x.
Recent outbreaks of listeriosis have emphasized the urgent need for rapid and reliable detection methods for Listeria spp., especially in food. Haemolysin production is a major factor in the pathogenesis of listeriosis and the polymerase chain reaction (PCR) was used to amplify two specific DNA fragments of the alpha- and the beta-haemolysin genes. The amplification system specifically recognized L. monocytogenes strains. The detection limit determined with pure cultures was 10 bacteria when estimated with alpha-haemolysin primers. In the analysis of 50 samples of cooked sausage products, bacterial colonies suspected to be Listeria spp. were isolated by conventional methods from six samples. PCR analysis identified three of six as L. monocytogenes. Subsequent serotyping showed perfect agreement with the PCR results. Since enrichment is the most time consuming step in conventional methods a PCR procedure which allows the direct detection of L. monocytogenes in milk was developed. Pasteurized milk was artificially contaminated with various levels of L. monocytogenes. The detection limit was determined to be 10 bacteria/10 ml milk and direct detection and identification of L. monocytogenes took less than two working days. These results show that this haemolysin gene amplification system is very rapid and reliable and therefore avoids cumbersome and lengthy cultivation steps.
近期李斯特菌病的爆发凸显了对李斯特菌属快速可靠检测方法的迫切需求，尤其是在食品检测方面。溶血素的产生是李斯特菌病发病机制中的一个主要因素，聚合酶链反应（PCR）被用于扩增α-溶血素基因和β-溶血素基因的两个特定DNA片段。该扩增系统能特异性识别产单核细胞李斯特菌菌株。用α-溶血素引物评估时，纯培养物的检测限为10个细菌。在对50份熟香肠产品样本的分析中，通过传统方法从6个样本中分离出疑似李斯特菌属的菌落。PCR分析确定6个样本中的3个为产单核细胞李斯特菌。随后的血清分型结果与PCR结果完全一致。由于富集是传统方法中最耗时的步骤，因此开发了一种可直接检测牛奶中产单核细胞李斯特菌的PCR方法。用不同水平的产单核细胞李斯特菌人工污染巴氏杀菌牛奶。检测限确定为每10毫升牛奶中有10个细菌，直接检测和鉴定产单核细胞李斯特菌耗时不到两个工作日。这些结果表明，这种溶血素基因扩增系统非常快速可靠，因此避免了繁琐冗长的培养步骤。
