实时荧光定量PCR检测汉赛巴通体

目的采用新型TaqMan-MGB探针建立检测汉赛巴通体的实时荧光定量PCR方法。方法根据汉赛巴通体特异的16S-23S rRNA间隔区序列设计引物和探针,以克隆的16S～23S rRNA间隔区基因片段作DNA模板,在荧光定量PCR检测仪(ABI 7900HT)上建立实时荧光定量检测方法,对所建立的方法分别进行敏感性、特异性及重复性分析,并且对模拟标本进行检测。结果建立的定量标准曲线的循环阈值(C_(?))与模板拷贝数呈良好的线性关系(r=0.997);与普通PCR相比较,荧光定量PCR检测的灵敏度是其1000倍。用荧光定量PCR检测其他相关立克次体和细菌DNA样本,除军团菌检出微弱信号(2个拷贝)外,其余检出结果均为0;对重复性进行了评价,批内和批间的变异系数在0.2%～1.9%之间。用荧光定量PCR检测汉赛巴通体感染的小鼠血标本,在感染后第2天、第3天、第5天,检出少量的汉赛巴通体,在感染后的第1天和第2天从脾脏样本中检出大量的汉赛巴通体。结论检测汉赛巴通体实时荧光定量PCR方法具有高度特异性和高敏感性以及良好的重复性,可用于快速检测各种样本中的微量汉赛巴通体以及作为汉赛巴通体感染的实验室诊断。

Development of a quantitative real-time polymerase chain reaction for detecting Bartonella henselae

Objective To develop a quantitative real-time polymerase chain reaction (PCR) for detecting Bartonella henselae.Methods According to the 16S-23S rRNA intervening sequences(IVS) specific for B.henselae,one pair of primers and one TaqMan-MGB probe were designed.A quantitative real-time PCR was developed with the primers,the probe,and the IVS,a standard template,in DNA sequence detection system(ABI 7900HT).Results The standard curve was established with the standard template and the relationship between the value of threshold cycle(C_(?)) and the DNA copy number was linear(r=0.997).The sensitivity of this quantitative real-time PCR was about 1000 times higher than that of a common PCR used to detect homologous DNA.By this quantitative real-time PCR,the DNA sample of B.henselae was positively detected but not from other rickettsial or bacterial DNA samples.The variation coefficients of intra- and inter-assay reproducibility were 0.2%-1.9%.Using the real-time quantitative PCR to detect samples from mice that were experimentally infected with B.henselae,the small amount of B.henselae DNA was detected in blood samples on days 2,3,and 5 and large amount of B.henselae DNA was detected in spleen samples on days 1 and 2 after infection.Conclusion Results from our study suggested that this quantitative real-time PCR was highly specific,sensitive and with good repeatability for detection of B.henselae.It seemed quite useful for rapid detection of tiny DNA of B.henselae in various samples and laboratory diagnosis of bartonellosis caused by B.henselae.