Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d'Etudes et de Recherches sur la Qualité des Aliments et les Procédés Agro-alimentaires, Maisons-Alfort, France.
J Appl Microbiol. 2009 Aug;107(2):465-73. doi: 10.1111/j.1365-2672.2009.04215.x. Epub 2009 Mar 9.
To develop real-time PCR assays for tracking and tracing clostridia responsible for human botulism.
Real-time PCR assays based on the detection of the genes ntnh encoding the nontoxin-nonhaemagglutinin (NTNH) proteins or the most homologous regions of the botulinum neurotoxin (bont) genes have been developed together with four real-time PCR assays, each being specific of the genes bont/A, bont/B, bont/E, bont/F and enables a toxin type-specific identification. The specificity of the assays was demonstrated using a panel of botulinum toxin producing clostridia (29 strains), nonbotulinum toxin producing clostridia (21 strains) and various other bacterial strains. The toxin type-specific assays had a sensitivity of 100 fg-1000 fg of total DNA in the PCR tube (25-250 genome equivalents) which correspond to 10(3) to 10(4) cells ml(-1). After a 48 h enrichment in anaerobic conditions, these PCR assays allowed the detection of Clostridium botulinum type A in a naturally contaminated sample of 'foie gras' suspected in a C. botulinum outbreak.
These PCR tests are specific and reliable for detection of heterogeneous BoNT producing clostridia responsible for human botulism.
Adoption of these PCR assays is a step forward a reliable and rapid detection of these clostridia in food samples.
开发实时 PCR 检测方法，用于追踪和溯源引起人类肉毒中毒的梭菌。
基于检测编码非毒素-非血凝素（NTNH）蛋白的 ntnh 基因或肉毒神经毒素（bont）基因最同源区域，我们开发了实时 PCR 检测方法，同时还开发了 4 种实时 PCR 检测方法，每种方法均针对 bont/A、bont/B、bont/E、bont/F 基因具有特异性，可实现毒素类型特异性鉴定。采用一组产肉毒梭菌（29 株）、非产肉毒梭菌（21 株）和其他各种细菌菌株对检测方法的特异性进行了验证。毒素类型特异性检测方法在 PCR 管中总 DNA 的灵敏度为 100 fg-1000 fg（25-250 个基因组当量），相当于 10(3)至 10(4)个细胞/ml(-1)。经过 48 小时在厌氧条件下的富集，这些 PCR 检测方法能够在疑似肉毒梭菌爆发的“鹅肝酱”自然污染样本中检测到 A 型肉毒梭菌。
这些 PCR 检测方法对检测引起人类肉毒中毒的异质性 BoNT 产生梭菌具有特异性和可靠性。
采用这些 PCR 检测方法是在食品样本中可靠、快速检测这些梭菌的重要一步。
