目的 确定不同梅毒螺旋体TpN47基因序列的差异性及其跨膜结构,建立基于TpN47基因检测梅毒螺旋体的荧光定量PCR方法.方法 通过使用NCBI/Blast,Pfam,TMHMM Server v.2.0等网络资源分析梅毒螺旋体TpN47基因编码蛋白的保守功能结构域和跨膜区域.根据参考序列设计引物,采用PCR技术从梅毒螺旋体基因组DNA中扩增TpN47基因,将其连接入克隆载体pMDl8-T进行测序,并用DNASTAR软件比较不同梅毒螺旋体菌株TpN47基因的核苷酸序列.根据梅毒螺旋体TpN47基因保守区域设计引物,建立基干该目的 基因的荧光定量分析PCR检测方法并分析其灵敏度、特异性和稳定性.同时,采用基于TpN47基因的荧光定量PCR对梅毒螺旋体感染患者血液样本进行检测.结果 研究表明,梅毒螺旋体TpN47基因为梅毒螺旋体外膜蛋白编码基因,其产物定位干外膜表面.不同梅毒螺旋体菌株中TpN47基因的核苷酸序列保守,相似度达99%以上.基于TpN47基因的荧光定量PCR方法可有效地检测梅毒螺旋体感染患者血清及泌尿道分泌物样本中的梅毒螺旋体.结论TpN47基因为梅毒螺旋体外膜蛋白编码基因,建立的基于TpN47基因的荧光定量PCR方法具有快速、敏感、稳定等优点,适用于梅毒螺旋体临床实验室诊断.%To establish a real time quantitative PCR to detect T. Pallidum based on outer membrane protein coding gene (TpN47) , the structure domains and transmembrane regions were analyzed through on line database of NCBI, Pfam and TM HMM Server v. 2. 0. The primers were worked out according to the DNA sequence of TpN47. The TpN47 gene was amplified from the total cell DNA of T. Pallidum by PCR, and then inserted into the cloning vector pMD18 T. The sequence of inserted fragment was sequenced, and the T. Pallidum sequences from different T. Pallidum strains were compared through DNAS TAR. Quantitative PCR assay based on TpN47 gene for quick detection of T. Pallidum in serum from people infected by T. Pallidum was conducted. The prediction result suggested that TpN47 located at the outside of outer membrane. The similari ties of nucleotide sequences of TpN47 genes from different T. Pallidum strains were above 99%. The detection result of real time fluorescent quantitative PCR were positive for all the specimens of serum collected from the T. Pallidum infected people. The conserved TpN47 genes were presented in different strains of T. Pallidum. In concluson the real time fluorescent quanti tative PCR has been established for detecting T. Pallidum and its advantages including quickness, stability, sensitivity and specificity, which indicates that this method could be used for clinical laboratory diagnosis of T. Pallidum infection.
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