摘要
目的:利用荧光定量PCR技术,建立一种快速、敏感、特异的检测产气荚膜梭菌的方法,及时用于指导临床治疗。方法:以产气荚膜梭菌16srRNA基因作为靶序列,设计一对特异引物和探针,以伤口分泌物和脓液提取的核酸作为模板,利用已经优化的引物和探针进行PCR反应;同时与细菌培养作比较,验证此方法的快速性、敏感性及特异性。结果:建立的反应体系在上游引物浓度为0.45μmol/L,下游引物浓度为0.15μmol/L,探针浓度为0.3μmol/L时,具有很好的敏感性,与21种其他细菌均无交叉反应,其敏感性为9cfu/反应体系。荧光定量PCR检测结果与细菌培养结果完全一致。结论:所建立的荧光定量PCR方法特异、灵敏、快速,能对产气荚膜梭菌感染做出准确的检测报告,具有对战时高发疾病气性坏疽进行快速和定量检测潜质。
Objective：To establish a rapid,sensitive and specific real-time PCR method for detection of Clostridium perfringens（C.perf）and application in clinic.Method：The C.perf-specific primers and probe were designed through a specific primer designing program based on C.perf-specific sequence,DNA of secretion and liquor puris were tested with this assay.To confirm sensitivity,speedability and specificity of this method compared with bacterial culture.Results：The best forward and reverse primers concentration were 0.45μmol/L and 0.15μmol/L respectively,the best probe concentration was 0.3μmol/L.This technique had good sensitivity,had no consensual reaction with other 21 bacterium species.The result of PCR accorded with that of the culture.Conclution：.The Taqman PCR assay,especially in the war,can be used for the rapid,sensitive and specific detection and enumeration of Clostridium perfringens from secretion and liquor puris with a short enrichment.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2010年第12期72-75,共4页
China Biotechnology
基金
全军医学科学技术研究"十一五"专项(08Z009)资助项目
