百日咳杆菌荧光定量PCR检测方法的建立和应用  

Development of fluorescence quantitative PCR for detection of Bordetella pertussis and its application

作　　者：徐颖华[1] 徐运强[2] 张庶民[1] 王丽婵[1] 侯启明[1] 雷殿良[1] 

机构地区：[1]中国药品生物制品检定所血清室,北京100050 [2]河北省石家庄市第二医院检验科

出　　处：《中华检验医学杂志》2008年第6期690-694,共5页Chinese Journal of Laboratory Medicine

摘　　要：目的建立快速、准确、特异的定量检测百日咳杆菌的方法，并进行临床应用研究。方法根据百日咳杆菌IS481基因序列，设计并合成引物和荧光探针，建立百日咳杆菌荧光定量PCR检测方法，并对方法的特异度、重复性和灵敏度进行评价，检测百日咳疑似患者咽拭子225份和健康者咽拭子30份。结果该方法的标准曲线显示模板浓度在10^2-10^8拷贝／μl线性范围与其循环阈值（Ct）具有较好的线性关系，相关系数（r）为0．998，最小检出量为10^2拷贝。特异度较好，灵敏度较高，重复性实验结果显示在同一时间检测高低浓度阳性克隆质粒（2×10^7与3×10^3拷贝／μ1）10次，连续检测5次，变异系数（CV）为5．78％-16．7％。在不同时间检测高低浓度阳性克隆质粒（2×10^7与3×10^3拷贝／μl），连续7次，CV为8．25％～14．9％。经检测，疑似患者的咽拭子有41份样本为阳性，30份健康人咽拭子均为阴性。结论由于本实验建立的荧光定量PCR方法快速、灵敏度高、特异度好，适合临床实验室进行临床标本的百日咳杆菌的定量检测，有较大的应用前景。Objective To establish a rapid, accurate, specific quantitative assay for detecting B. pertussis, and apply to clinical diagnosis. Methods According to the specific sequence of B. pertussis IS481 gene, the primers and the fluorescence probe were designed and synthesized. Then a fluorescence quantitative PCR for detecting B. pertussis was developed. The specificity, sensitivity and reproducibility of the method were evaluated. 255 specimens including 225 nasopharyngeal swabs from suspected pertussis patients and 30 normal nasopharyngeal swabs were detected by fluorescence quantitative PCR. Results A rapid specific quantitative method for detecting B. pertussis was established. The standard curve of the method indicated that there was a good linear relationship between the CT value and the template concentration with the correlation coefficient being 0. 998. The linear range of the system was from 10^2 to 10^8 copies/μl and the minimum was 10^2 copies. It had a high sensitivity and good specificity. The intra-and inter-assay coefficients of variation were 5.78% -16.7% and 8.25% -14.9% respectively. The fluorescence quantitative PCR identified 41 positive results for specimens from suspected pertussis patients and results of 30 normal specimens were all negative. Conclusions The method can quantitatively detect the B. pertussis rapidly with high sensitivity and specificity, it can be applied to clinical diagnosis.

关 键 词：百日咳 咽 博德特菌 百日咳 聚合酶链反应 

分 类 号：R686[医药卫生—骨科学]