海南省澄迈县斑点热群立克次体分离株多间隔序列分型

Multispacer typing of spotted fever group rickettsiae isolated from Chengmai county of Hainan province, China

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 Spotted fever caused by spotted fever group rickettsiae （SFGR） is found throughout China. During 2007-2008, 28 human SFGR isolates and 34 rat SFGR isolates （including 15 isolates from Rattus fulvescens, 5 isolates from R. edwardsi, 7 isolates from Callosciurus erythraeus roberti, and 7 isolates from Dremomys rufigenis） were gathered by L929 cell culture. Previous research indicated that these isolates shared identical sequences of 16S rRNA,gltA, ompA, groEL, and 17×103 protein gene with each other, and were of the same serophenotype. This project aims to study the 62 SFGR isolates from fever patients and wild rodents in Chengmai county of Hainan province, China by multispacer typing （MST）.Methods Nine pairs of primers which had been previously reported in MST of SFGR were used in amplification and sequencing of the 62 isolates in this study. Sequences were subjected to Blast analysis after data correction. Analysis of single nucleotide polymorphisms （SNPs） was performed with Megalign. Six spaced sequences that were successfully sequenced from each isolate were spliced and applied in the construction of phylogenetic tree. Results There were great variations among the 62 isolates. A total of 48 mutant genotypes were identified. The predominant genotype, h3, accounted for 21.7% （13/60） of all the isolates tested, and the remaining 47 genotypes were all unique, each accounting for 1.6% （1/60） of all isolates. Phylogenetic analysis showed that all these 48 genotypes were classified in the same cluster. Among all reference strains included in this study, R. heilongjiangensiswas genetically closest to these isolates. Thymine-to-cytosine transitions had the highest percentage （52/114, 45.6%） among all SNPs, followed by adenine-to-guanine transitions （37/114, 32.5%）.Conclusion The non-coding spaced sequences of SFGR prevailing in Chengmai county of Hainan province are highly heterologous. The selected primers for MST of SFGR are capable of detecting rickettsial pathogens. MST can be used in the rapid and effective tracing of endemic strains in outbreak of rickettsiae.