实时荧光定量PCR快速检测百日咳鲍特菌方法学建立及评价  

Development and evaluation of real-time quantitative PCR assay for rapidly detecting Bordetella pertussis

作　　者：王慧[1] 罗炜[2] 赖克方[2] 

机构地区：[1]广州医学院第一附属医院检验科,广州510120 [2]广州医学院第一附属医院呼吸疾病国家重点实验室,广州510120

出　　处：《临床检验杂志》2010年第6期407-409,共3页Chinese Journal of Clinical Laboratory Science

基　　金：广州市科技攻关重点项目基金(2002Z2-E0091);广东省科技计划项目基金(2010B031600145)

摘　　要：目的建立实时荧光定量PCR检测百日咳鲍特菌的方法。方法以百日咳鲍特菌的重复插入序列IS481为目的基因,设计探针和引物,以克隆的IS481基因片段为DNA模板,在荧光定量PCR仪上建立实时荧光定量PCR检测方法和标准曲线,并进行灵敏度、重复性、特异性实验。结果建立的定量标准曲线阈值循环数(Ct)与模板拷贝数呈良好线性关系(r=0.998);最低检测浓度为102 copies/μl;批内及批间变异系数均小于4%;对临床其他常见呼吸道病原体不出现特异性扩增曲线。结论实时荧光定量PCR法检测IS481基因可用于百日咳鲍特菌的快速检测,具有较好的灵敏度、特异性及重复性。Objective To establish a method to detect Bordetella pertussis based on real-time fluorescent quantitative PCR.Methods The primers and Taqman probe were designed according to the repetitive insertion sequence IS481 of Bordetella pertussis.The DNA fragment of IS481 gene of Bordetella pertussis amplified by PCR was used as the template to develop the assay and standard curve.The sensitivity,specificity and reproducibility of the developed real-time quantitative PCR were tested.Results A fine linear relationship（r =0.998） between the threshold cycle（Ct） values of the developed standard curve and the copy number of template was observed.The sensitivity of the assay was 102 copies/μl and the coefficients of variation of intra and inter-assay were less than 4%.No amplication curve of other respiratory pathogens was found thus perfect specificity was shown.Conclusions A real-time fluorescent quantitative PCR for detecting the repetitive element IS481 was successfully developed,and the approach should be available for rapid diagnosis of Bordetella pertussis with good sensitivity,specificity and reproducibility.

关 键 词：百日咳鲍特菌 聚合酶链反应 实时荧光定量 IS481基因 

分 类 号：R516.6[医药卫生—内科学] Q503[医药卫生—临床医学]