山东地区恙虫病东方体分离株的基因分型与序列分析

Genotype identification and sequence analysis of Orientia tsutsugamushi isolated from Shandong area

目的鉴定山东地区恙虫病东方体分离株的基因型别,分析其核苷酸序列。方法以日本、韩国常用的恙虫病东方体分型引物,用巢式PCR技术检测恙虫病东方体分离株的相对分子质量(Mr)56×103蛋白基因片段,对群引物扩增的PCR产物纯化回收后测序,并行序列分析。结果Gilliam、Karp、Kato、山东分离株用群引物均扩增出481-507 bp的目的片段,Gilliam、Karp、Kato经相应的型引物扩增出了目的片段。山东分离株仅Kawasaki型的分型引物扩增出目的条带,序列分析证实山东分离株与Kawasaki型相似。结论在国内首次扩增出Gilliam、Karp、Kato三型标准株,山东地区恙虫病东方体分离株为Kawasaki型;山东地区恙虫病东方体与日本的Kawasaki型相似,两者有较近的亲缘关系。

Objective To determine genotype, nucleotide sequence homology and phylogenesis of Orientia tsutsugamushi isolated from Shandong, China. Methods Orientia tsutsugamushi isolated from patients, Apodemus agrarius and Leptotrombidium scutellare in Shandong area were identified by nested PCR. On the basis of the nucleotide sequence of the gene that encoding the Ot Mr 56 × 10^3 antigen, the primers were frequently used in Japan and Korea. Nucleotide sequences of three isolates were determined. The DNA sequences were compared with nucleotide sequences of Orientia tsutsugamushi registered in GenBank for sequence homology analysis. Phylogenetic analysis of the isolates and some published sequences was carried out with Neighbor-joining method by MEGA 3.1 software. Results 481- 507 bp DNA fragments encoding Orientia tsutsugamushi M, 56 × 10^3 protein were amplified successfully in the samples of Gilliam, Karp, Kato and Shandong isolates by group-specific primers. The corresponding target fragments of the three international reference strains of Gilliam, Karp, and Kato were amplified successfully with each of their own type specific primers. 523 bp DNA fragments were amplified successfully from Shandong isolates by the nPCR with Kawasaki-specific primer, and no DNA fragment was amplified by the nPCR with Gilliam, Karp, Kato, Kuroki and Saitama-specific primer. Comparing with the sequences of Orientia tsutsugamushi registered in GenBank, all the Shandong isolates shared higher than 95% nucleotide sequence homology with Kawasaki strain founded in japan. Data from phylogenetic analysis showed that Shandong isolates belonged to the same branch with Kawasaki strain. Conclusion To facilitate international comparison and communication, the primers should be employed in the Orientia tsutsugamushi research in China. Orientia tsutsugarnushi isolated in China were similar to Kawasaki strain in japan.