摘要
目的建立特异、敏感、快速检测CAR菌的TaqMan MGB探针荧光定量PCR方法。方法针对CAR菌16S rRNA基因序列设计特异性引物和探针,建立MGB探针荧光定量PCR方法,验证方法的特异性、敏感性和稳定性。对2008～2012年期间采集的1 344份临床标本中的CAR菌进行检测,同时进行普通PCR检测作为对照。结果 CAR菌TaqMan MGB探针荧光定量PCR方法具有高度特异性,与肺炎支原体、侵肺巴斯德菌、支气管鲍特杆菌、肺炎克雷伯杆菌、大肠埃希菌、肺炎链球菌间无交叉反应,检测灵敏度达2.7拷贝。标准曲线显示各浓度范围内具有良好线性关系,相关系数为0.999,斜率为-3.22,TaqMan MGB探针荧光定量PCR效率为104.432%。对1 344份标本进行检测,结果 TaqMan MGB探针荧光定量PCR检出510份CAR菌阳性样本。TaqMan MGB探针荧光定量PCR能够直接从标本中检出CAR菌DNA,检测时间仅为40 min。结论 TaqMan MGB探针荧光定量PCR方法具有可靠、特异、敏感的特点,适用于CAR菌的快速检测。
Objective To establish a TaqMan MGB probe-based, sensitive and specific real-time fluorescence quantitative PCR assay for rapid detection of cilia-assoclated respiratory （ CAR ） bacillus. Methods Primers and probes specific to 16S ribosomal RNA （16S rRNA）gene sequence of cilia-associated respiratory bacillus were designed. A TaqMan MGB probe-based, real-time fluorescence quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed. Then, the established TaqMan MGB probe-based real-time fluorescence quantitative PCR assay was applied to detect cilia-associated respiratory bacillus in 1344 clinical specimens during 2008 - 2012, and compared with conventional PCR assay. Results The specificity of this established TaqMan MGB probe-based real-time fluorescence quantitative PCB assay was high for detecting cilia-associated respiratory bacillus, and there were no cross-reactivity with Mycoplasma pneumoniae , Pasteurella pneumotropica , Bordetella bronchiseptica , Klebsiella pneumoniae , Escherichia coli and Streptococcus pneumoniae. The detection limits was 2. 7 copies. The correlation coefficient and slope value of standard curve were 0. 999 and -3.22, respectively, and the efficiency of TaqMan MGB-hased probe-based real-time fluorescence quantitative PCR assay was 104. 432%. The TaqMan MGB-based probe real-time fluorescence quantitative PCR assay was preformed to detect cilia-associated respiratory bacillus in 1344 clinical specimens. A total of 510 specimens were positive for cilia-associated respiratory bacillus. This established PCR technique could detect cilia-associated respiratory bacillus DNA from clinical specimens directly, and the detection time was only 40 minutes needed, Colusion The TaqMan MGB-based probe-based real-time fluorescence quantitative PCR assay established in this study is a reliable, specific, sensitive and useful tool for rapid detection of cilia-associated respiratory bacillus.
出处
《中国比较医学杂志》
CAS
2013年第8期9-13,19,共6页
Chinese Journal of Comparative Medicine
关键词
CAR菌
16S
RRNA基因
小沟结合物探针
实时荧光定量PCR
快速检测
Cilia-associated respiratory （CAR） bacillus
16S ribosomal RNA gene
Minor groove binder probe
Real-time fluorescence quantitative PCR
Rapid detection
