| 杨晓,陈梅玲,温博海,牛东升,朱丽娜,李青凤,孙长俭.实时荧光定量PCR检测普氏立克次体[J].Chinese journal of Epidemiology,2006,27(11):963-967 |
| 实时荧光定量PCR检测普氏立克次体 |
| Detection of Rickettsia prowazekii by quantitative real-time PCR3 |
| Received:March 06, 2006 |
| DOI： |
| KeyWord: 流行性斑疹伤寒 普氏立克次体 实时定量PCR 外膜蛋白B基因 |
| English Key Word: Epidemic typhus Rickettsia prowazekii Quantitative real-time PCR ompB |
| FundProject:国家科技攻关课题资助项目(2003BA712A04-07) |
| Author Name | Affiliation | E-mail | | YANG Xiao | Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China | | | CHEN Mei-ling | Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China | | | WEN Bo-hai | Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China | bohaiwen@sohu.com | | NIU Dong-sheng | Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China | | | ZHU Li-na | Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China | | | LI Qing-feng | Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China | | | SUN Chang-jian | Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China | |
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| Abstract: |
| 目的采用新型TaqMan-MGB探针建立检测普氏立克次体的实时荧光定量PCR方法。方法根据普氏立克次体外膜蛋白B的基因(ompB)序列设计引物和探针,以克隆的OmpB基因片段作DNA模板,在荧光定量PCR检测仪(ABI 7900型)上建立实时荧光定量检测方法。结果建立的定量标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0．999)；与巢式PCR相比较,荧光定量PCR检测敏感性是其100倍。用荧光定量PCR检测莫氏立克次体及其他相关立克次体和细菌DNA,检出结果均为阴性。用荧光定量PCR检测普氏立克次体感染的豚鼠血标本,某些样本检测为阳性,而用巢式PCR检测的结果均为阴性。结论研究中建立的检测普氏立克次体实时荧光定量PCR具有很高的特异性和敏感性,适合于快速检测样本中微量普氏立克次体DNA,可用作临床实验室快速确诊流行性斑疹伤寒。 |
| English Abstract: |
| Objective To develop a quantitative real-time polymerase chain reaction (PCR) for detecting Rickettsia prowazekii.Methods Primers and TaqMan-MGB probes designed based on ompB gene of R.prowazekii,were used to develop this method.Results For the quantitative real-time PCR, the relationship between the values of threshold cycle (Ct) and the DNA copy number was linear (r= 0.999) and the sensitivity was about 100 times higher than that of the nested PCR for detecting the same DNA sample.The results of the genomic DNA samples of other rickettsial and bacterial agents detected by real-time PCR were all negative.DNAs extracted from blood samples of guinea pig infected with R.prowazekii were examined by real-time PCR and the positive results were obtained from some of these samples.However,the results of some samples in nested PCR assay were all negative.Conclusion These results suggested that the real-time PCR was highly specific and sensitive for detection of R.prowazekii that was useful for the detection of tiny DNA of R.prowazekii in blood samples from patients suspected of having epidemic typhus. |
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