检测贝氏柯克斯体的实时荧光定量PCR

目的 采用新型TaqMan-MGB探针建立检测贝氏柯克斯体的实时荧光定量PCR（real—time quantitative PCR）方法。方法 根据贝氏柯克斯体特异的23S rRNA插入基因序列设计引物和探针，以克隆的23S rRNA插入基因片段作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=0．997）；与套式PCR相比较，荧光定量PCR检测敏感性是其100倍。用荧光定量PCR检测其它相关立克次体，检出结果均为0。用荧光定量PCR检测贝氏柯克斯体感染的小鼠脾脏标本，脾脏中贝氏柯克斯体的感染量与感染过程具有相关性。结论 本研究建立的检测贝氏柯克斯体的实时荧光定量PCR具有很高的特异性和敏感性。特别适合样本中微量贝氏柯克斯体DNA的检测，其定量检测可用于动物实验中的贝氏柯克斯体感染程度的分析。

Detection of Coxiella burnetii by real-time quantitative PCR

According to the 23S rRNA intervening sequences (IVS) specific for Coxiella burnetii, a pair of primers and one TaqMan-MGB probe were designed. A real-time quantitative polymerase chain reaction (PCR) was developed with the primers, the probe, and the template (23S IVS DNA fragment of C.burnetii). The standard curve was established with the 23S IVS DNA in DNA sequence detection system (ABI 7900HT) and the relationship between the values of threshold cycle (Ct) and the DNA copy number was linear (r=0.997), and the sensitivity of this real-time quantitative PCR was about 100 times higher than that of the nested PCR used to detect homologous DNA. The genomic DNA samples of other rickettsial agents were detected by this real-time quantitative PCR and the results were all negative. Using the real-time quantitative PCR to analyze the spleen sample from the mice infected with C. burnetii, the number of the infectious agent was correlated with the progression of the infection. These results suggest that the real-time quantitative PCR is highly specific and sensitive for detection of C.burnetii. It is very useful for detection of tiny DNA of C. burnetii in samples and for analysis the progression of animals experimentally infected with C. burnetii.