Letertre Capucine, Perelle Sylvie, Dilasser Françoise, Fach Patrick
Unité: Atelier de Biotechnologie, Laboratoire d'Etudes et de Recherches sur l'Hygiène et la Qualité des Aliments, Agence Française de Sécurité Sanitaire des Aliments (AFSSA), 1-5 rue de Belfort, 94700, Maisons-Alfort, France.
Mol Cell Probes. 2003 Aug;17(4):139-47. doi: 10.1016/s0890-8508(03)00045-8.
This paper describes real-time fluorescence PCR assays for detecting and toxinotyping nine enterotoxin genes from Staphylococcus aureus. A universal set of primers allowed sea, seb, sec, sed, see, seg, seh, sei, sej enterotoxin genes from S. aureus to be detected in a single real-time PCR assay with the LightCycler (LC) instrument. Using the universal forward primer and a type-specific reverse primer, real-time PCR assays allowed the S. aureus enterotoxin genes to be specifically genotyped. A collection of S. aureus isolates (n=83) was detected and further characterised for sea, seb, sec, sed, see, seg, seh, sei, sej, using real-time PCR assays, and data were compared with those obtained by conventional block cycler PCR. Isolates were also tested for their ability to produce staphylococcal enterotoxins A, B, C and D by a commercial reversed passive latex agglutination (RPLA) test. Real-time PCR assays developed on the LightCycler system (LC-PCR) are a powerful tool for rapid detection and toxinotyping of the enterotoxin genes sea to sej from S. aureus. The work offers a very quick, reliable and specific alternative to conventional block cycler PCR assays to identify the enterotoxin profile of toxigenic S. aureus.
本文描述了用于检测金黄色葡萄球菌9种肠毒素基因并进行毒素分型的实时荧光PCR检测方法。一套通用引物可使金黄色葡萄球菌的sea、seb、sec、sed、see、seg、seh、sei、sej肠毒素基因在使用LightCycler（LC）仪器的单一实时PCR检测中被检测出来。使用通用正向引物和型特异性反向引物，实时PCR检测可对金黄色葡萄球菌肠毒素基因进行特异性基因分型。利用实时PCR检测方法对一组金黄色葡萄球菌分离株（n = 83）进行检测，并进一步对其sea、seb、sec、sed、see、seg、seh、sei、sej基因进行特征分析，同时将数据与通过传统块循环PCR获得的数据进行比较。还通过商业反向被动乳胶凝集（RPLA）试验检测分离株产生葡萄球菌肠毒素A、B、C和D的能力。在LightCycler系统上开发的实时PCR检测方法（LC-PCR）是快速检测金黄色葡萄球菌肠毒素基因sea至sej并进行毒素分型的有力工具。这项工作为鉴定产毒素金黄色葡萄球菌的肠毒素谱提供了一种非常快速、可靠且特异的替代传统块循环PCR检测的方法。
