巢式PCR及序列分析用于攀枝花市恙虫病东方体检测

Detection of Orientia tsutsugamushi in Panzhihua city by SN-PCR and sequence analysis

目的对采集自攀枝花市的疑似恙虫病患者血液样本进行实验室检测,了解攀枝花地区恙虫病东方体感染情况,同时为四川省恙虫病早期诊断提供依据。方法运用巢式PCR对2013年7-10月采集自攀枝花市的48份疑似恙虫病患者全血样本中提取的基因组DNA进行恙虫病东方体sta-58编码基因检测;随机挑取2份阳性样本进行恙虫病东方体sta56基因片段扩增、测序及序列分析。结果 48份样本中,巢式PCR检测阳性的样本18份;2份样本均扩增到恙虫病东方体sta56基因片段,其序列同源性为96%,系统进化分析显示该2株恙虫病东方体均与某些恙虫病东方体台湾株位于同一个分支,同时与Karp株位于同一个分支。结论在四川省范围内首次运用巢式PCR法检测恙虫病东方体,实验室确诊攀枝花市存在恙虫病的感染,攀枝花市部分恙虫病患者由恙虫病东方体Karp型感染,在遗传进化上与某些台湾恙虫病东方体株关系密切。

Objective To detect Orientia tsutsugamushi in the blood specimens from suspect patients and get a clear understanding of the infection status in Panzhihua, Sichuan province. Methods The whole bacterial genomic DNA were extracted from 48 patients＇ blood specimens, which were collected from the city of Panzhihua during July to October in 2013. The sta-58 gene of O. tsutsugamushi was detected by the nested PCR （SN-PCR）. The partial sta56 genes were amplified, sequenced and analyzed from two cases of SN-PCR positive specimens selected randomly. Results There were 18 SN-PCR positive specimens deteced from 48 blood specimens. The partial sta56 genes were amplified successfully from two specimens and the sequences homology was 96%. Phylogenetic analysis showed that the Panzhihua43, Panzhihua45 and some Taiwan O. tsutsugamushi Strains were in the same branch, meanwhile, in the same branch with the O. tsutsugamushi Karp as well. Conclusion We have firstly detected the O. tsutsugamushi in the blood specimens by SN-PCR in Sichuan province. The existing infection of O. tsutsugamushi in Panzhihua was confirmed by the laboratory detection. Our study indicated that part of O. tsutsugamushi disease patients in Panzhihua were infected by Karp serotype, and the O. tsutsugamushi were closely related to some Taiwan O. tsutsugamushi Strains in genetic evolution.