摘要
目的利用荧光定量PCR技术,建立快速敏感特异的产气荚膜梭菌检测方法。方法以产气荚膜梭菌基因作为靶序列,设计一对引物和探针,以产气荚膜梭菌菌株提取核酸DNA作为模板,优化引物和探针的浓度比,同时验证方法的灵敏度、特异性、重复性。结果反应体系在上、下游引物浓度均为1μmol/L,探针浓度为0.1μmol/L时,具有良好的特异性和灵敏度,与创伤弧菌等24种相关细菌均无交叉反应,对纯菌检测的灵敏度为9×102CFU/ml,重复性试验证实该体系重复性好,稳定性高;3例临床样本检测结果与细菌培养结果相符。结论建立的实时荧光PCR方法特异、灵敏、快速,能对临床气性坏疽做出快速准确的报告,实现对气性坏疽战时高发性疾病安全、快速和定量检测。
OBJECTIVE To establish a rapid, sensitive and specific detection method of Clostridium perfringens with TaqMan PCR. METHODS A pair of primers and probe depending on gene were designed by way of target sequence of Clostridium perfringens, and apply clostridium perfringens of standard bacterium strain for template to appraise clostridium perfringens. The best primer and probe ratio were optimized. Specificity, sensitivity and stability analysis tests were performed by Clostridium perfringens and 24 other strains associated bacteria. RESULTS The best forward primers concentration was 1μM, reverse primer concentration was 1μM, and probe was 0.1μM. Test showed that the probe were highly conservative and specific. The results of all 24 strains bacteria were negative except of strains of Clostridium perfringens. The quantitative detection limit of the method was 9 × 10^2 CFU/ml. Repetitive experiments showed that this system had high stability and repeatability. The test results of 3 cases clinical samples were consistent with bacteria cultivation result. CONCLUSION The Real-time PCR method is of high sensitivity and specialty in the detection of Clostridium perfringens, which can be applied to the rapid detection of the bacteria in the balefulness disease.
出处
《中华医院感染学杂志》
CAS
CSCD
北大核心
2011年第13期2669-2673,共5页
Chinese Journal of Nosocomiology
基金
全军医学科学技术研究"十一五"专项(08Z009)
关键词
产气荚膜梭菌
探针
荧光定量PCR
Clostridium perfringens
TaqMan-based probe
Real-time PCR
