实时荧光定量PCR检测五日热巴通体

Detection of Bartonella quintana by a quantitative real-time polymerase chain reaction

目的采用TaqMan- MGB探针建立检测五日热巴通体的实时荧光定量PCR(real- timequantitativePCR)方法。方法根据五日热巴通体特异的16-23SrRNA间隔区序列设计引物和探针,以克隆的1623SrRNA间隔区基因片段作DNA模板,建立了检测五日热巴通体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=0.996);与普通PCR相比较,荧光定量PCR检测的灵敏度约为它的200倍。用荧光定量PCR检测其它相关立克次体和细菌,检出结果为阴性。实时荧光定量PCR检测五日热巴通体实验感染小鼠血、脾脏、肝脏和肺组织DNA样本,脾脏和肝脏样本中检出较高水平五日热巴通体DNA,血和肺部检出的目的DNA的水平较低。结论本研究建立的检测五日热巴通体的实时荧光定量PCR法具有很高的检测灵敏度和特异性,可用于临床患者血样本的检测,作五日热的早期诊断。

Aceording to the 16S-23S rRNA intervening sequences （IVS） of Bartonella quintana, one pair of primers and one TaqMan-MGB probe were designed. A quantitative real-time polymerase ehain reaetion （quantitative PCR） was developed with the primerS, the probe, and the template （16S-23S rRNA IVS DNA fragment of B. quintana）. The standard curve was established with the 16S-23S rRNA IVS DNA fragment and the relationship between the values of threshold cycle （Ct） and the DNA copy number was linear （r=0. 996）. The sensitivity of this quantitative PCR was about 200 times higher than that of a common PCR to detect the homologous DNA. In addition,this quantitative PCR was highly specific and sensitive for detection of B. quintana. It is very useful for detection of tiny DNA of B. quintana in samples for clinic diagnosis of bartonellosis.