Letertre Capucine, Perelle Sylvie, Dilasser Françoise, Fach Patrick
Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d'Etudes et de Recherches sur l'Hygiène et la Qualité des Aliments, Unité: Atelier de Biotechnologie, 1-5 rue de Belfort, Maisons-Alfort 94700, France.
Mol Cell Probes. 2003 Oct;17(5):227-35. doi: 10.1016/s0890-8508(03)00058-6.
We describe the development of a strategy based on 5' nuclease multiplex PCR for the rapid detection of nine enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej) of Staphylococcus aureus. The genotyping scheme consists in identifying these nine enterotoxin genes by three 5' nuclease Triplex-PCR assays. The strategy was evaluated using a collection of S. aureus reference strains previously examined with conventional PCR assays, and by testing previously characterized food S. aureus field strains. The 5' nuclease Triplex-PCR assays correctly detected the se genes in all the reference strains. In tests with field strains there was generally excellent agreement with the results obtained by conventional PCR, except for some strains harbouring variant se genes. The detection limits of the Triplex-PCR assays evaluated using fivefold dilution of recombinant plasmids for each se gene ranged from 16 to 2000 copies of target se genes in the PCR tube. The 5' nuclease Triplex-PCR assays developed are fast and specific, and provide a useful diagnostic tool for the detection and genotyping of se genes. The development of this method is an improvement that should facilitate epidemiological investigations of staphylococcal food poisoning outbreaks.
我们描述了一种基于5'核酸酶多重PCR的策略的开发，用于快速检测金黄色葡萄球菌的9种肠毒素基因（sea、seb、sec、sed、see、seg、seh、sei、sej）。基因分型方案包括通过三种5'核酸酶三重PCR检测来鉴定这9种肠毒素基因。该策略通过使用先前用常规PCR检测过的金黄色葡萄球菌参考菌株集合进行评估，并通过检测先前已鉴定特征的食品金黄色葡萄球菌现场菌株来进行测试。5'核酸酶三重PCR检测在所有参考菌株中均正确检测到了se基因。在对现场菌株的测试中，除了一些携带变异se基因的菌株外，与常规PCR获得的结果总体上具有很好的一致性。使用每种se基因的重组质粒五倍稀释液评估的三重PCR检测的检测限在PCR管中为目标se基因的16至2000拷贝。所开发的5'核酸酶三重PCR检测快速且特异，为se基因的检测和基因分型提供了一种有用的诊断工具。该方法的开发是一种改进，应有助于葡萄球菌食物中毒暴发的流行病学调查。
