用分子生物学技术诊断巴尔通体感染

Study on Bartonella infection using molecular biological diagnostic techniques from China

目的 应用聚合酶链反应(PCR)技术建立巴尔通体的检测鉴定方法并用于诊断临床疑似猫抓病合并双侧支气管肺炎患者。方法 根据16S～23S rRNA ITS基因序列较其他属细菌长,而且位于这段基因序列中的tRNA^(Ile)-tRNA^(Ala)基因间隔区具有高度变异性,tRNA^(Ile)和tRNA^(Ala)基因序列在巴尔通体属中完全保守,设计引物;同时应用文献发表的2对引物直接扩增1例临床可疑猫抓病患者全血基因组DNA。将研究设计引物的PCR产物直接测序,并进行序列分析。结果 3对引物扩增全血基因组DNA均获得巴尔通体特异基因片段,根据其中2对引物扩增产物大小与阳性对照不同,可初步确定样本与阳性对照菌株是不同巴尔通体;序列分析结果显示,研究设计引物TIle.455p-TAla.885n扩增产物核苷酸序列与中国云南省巴尔通体-分离株对应位置的序列相一致,同源性100％。结论 应用PCR技术从血液中直接扩增特异基因片段,可以快速检测巴尔通体感染;测序及序列分析结果进一步提供了此种致病巴尔通体在中国南北方均存在的线索。

Objective To establish polymerase chain reaction (PCR) technique for the detection of specific genes related to species of genus Bartonella , and for diagnosing clinically suspected cat-scratch disease (CSD) case complicated with pneumonia on both lungs. The appearance of Bartonella infectious diseases calls for genus and species detection and tools fOr identification in order to make clinical diagnosis and carry on epidemiological studies. Methods One pair of primer TIle. 455p-TAla. 885n was designed based on the fact that tRNA^(He)-tRNA^(Ala) intergenic spacer region in 16S-23S rRNA intergenic spacer(ITS) of genus Bartonella were high variable sequences flanked by completely conserved tRNA-encoding genes. 16S- 23S rRNA was longer than that which had been described in other bacteria. Two published pairs of primers were used to directly detect the specific gene fragments of Bartonella species DNA extracts from human blood, followed by PCR product Sequencing and nucleotide base sequence analysis. Results Amplification products of the three pairs of primers had the same predicted size of those in Bartonella spp. According to the different length of electrophoresis bank, the sample was identified as a species of genus Bartonella other than the positive control. Sequence analysis showed that the nuleotide sequence from the PCR product of primer TIle. 455p-TAla. 885n was identical to the Bartonella isolated from Yunnan in China. Conclusious PCR-based assay provided a simple and rapid means to detect pathogenic Bartonella species in humans and mammalian hosts as well as in arthropod vecters. This study suggested that this pathogenic Bartonella species existed in patients in northern and southern parts of China.