论文部分内容阅读
目的采用多间隔序列分型(MST)对从海南省澄迈县发热患者及野鼠分离的62株斑点热群立克次体进行分型研究。方法根据文献检索,应用9对成功用于斑点热群立克次体MST分型引物,对实验62株菌进行PCR扩增及测序。校正后序列进行Blast分析。同时,对序列进行Megalign比对分析单核苷酸多态性(SNPs)。将每株细菌成功测序的6段间隔序列按一定顺序拼接并构建进化树。结果使用6对间隔序列引物扩增实验菌株,结果显示每对引物扩增序列均显示较大的变异性,共获得48个变异型,以h3为代表的主要变异型占21.7%(13/60),剩余的47个变异型为独立型,分别占1.6%。进化树分析结果显示48个变异型均归为一簇。同其他参考菌株比较发现,黑龙江立克次体与上述菌株关系密切。SNPs分析发现位点变异主要以T-C转换及A-G转换为主。结论海南地区斑点热群立克次体分离株非编码间隔序列呈高度变异性。MST分型较传统的血清表型分型以及MLST分型更具有鉴别能力,对未来立克次体暴发流行可提供快速有效的流行株追踪。
Objective To identify 62 Rickettsia species of Rickettsia plague isolated from febrile patients and wild rats in Chengmai County, Hainan Province by multistep sequence typing (MST). Methods According to the literature search, nine pairs of MST genotyping primers were successfully used for speckle fever group Rickettsia, and 62 strains were amplified by PCR and sequenced. The corrected sequence is Blast analyzed. At the same time, the sequences were analyzed by Megalign single nucleotide polymorphisms (SNPs). The 6-interval sequence of successful sequencing of each strain of bacteria was spliced in a certain order and the phylogenetic tree was constructed. Results Six pairs of spacer sequences were used to amplify the experimental strains. The results showed that the amplified sequences of each primer showed great variability, a total of 48 variants were obtained, and the major variants represented by h3 accounted for 21.7% (13/60 ), The remaining 47 variants are independent, accounting for 1.6%. The results of phylogenetic tree analysis showed that all of the 48 variants were classified as a cluster. Compared with other reference strains, we found that Rickettsia in Heilongjiang is closely related to the above strains. SNPs analysis found that the site mutation mainly T-C conversion and A-G conversion. Conclusion The non-coding spacer sequences of Rickettsia plague in Hainan Province are highly variable. MST typing is more discriminatory than traditional serotyping and MLST typing, providing a rapid and effective follow-up of epidemic strains for future Rickettsia outbreaks.