中国嗜吞噬细胞无形体分离株多间隔序列分型分析

程雪琴; 张丽娟

Abstract

程雪琴,张丽娟.中国嗜吞噬细胞无形体分离株多间隔序列分型分析[J].Chinese journal of Epidemiology,2014,35(5):585-587

中国嗜吞噬细胞无形体分离株多间隔序列分型分析

Multispacer typing of Chinese Anaplasma phagocytophilum isolates

Received:December 31, 2013

DOI：10.3760/cma.j.issn.0254-6450.2014.05.026

KeyWord: 嗜吞噬细胞无形体多间隔序列分型

English Key Word: Anaplasma phagocytophilum Multispacer typing

FundProject:国家科技重大专项（2008ZX10004-008，2012ZX10004215）；国家基础研究项目—“973计划”（2010CB530206）

Author Name	Affiliation	E-mail
Cheng Xueqin	Department of Anaplasma, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Zhang Lijuan	Department of Anaplasma, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China	zhanglijuan@icdc.cn

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Abstract:

目的对我国嗜吞噬细胞无形体分离株进行多间隔序列分型（MST）分型。方法根据GenBank收录的4株无形体全基因组序列，利用Mauve 2.3.1软件行种内基因组比对，选择具有变异间隔区进行引物设计，通过引物特异性及扩增效率等预实验筛选引物，并对实验菌株进行PCR扩增，分析单核苷酸多态性（SNPs）。将每株菌间隔序列拼接后进行分型分析并构建进化树，分析不同地区、不同动物种类来源菌株遗传变异关系。结果共筛选出22对引物，用于我国11株无形体分离株 MST 分型，SNPs 分析显示，碱基转换发生率最高（60.2%，251/417），其中 A-G 转换居首（18.9%，79/417），颠换占23.0%（96/417），插入及缺失发生率占16.7%（70/417）。11株菌22条间隔序列拼接后（约11 047 bp）均为独立变异型。进化分析发现山东省莱州地区重症患者分离株（LZ-H1、H2、H3、H4、H5）及当地长角血蜱分离株 LZ-T1 与美国 Webster 株及 HZ 株聚为一簇，而北京分离株BJ-H1 与新疆分离株 XJ-H1 和 XJ-H3 聚为一类。莱州地区另一蜱分离株 LZ-T2 与新疆另外一人源分离株聚为一类，与莱州重症患者分离株遗传关系密切。结论我国无形体分离株间隔序列存在明显的遗传多态性，MST分型技术可用于无形体疫情暴发时的快速诊断和疫情追踪。

English Abstract:

Objective To type the Chinese Anaplasma phagocytophilum isolates by Multispacer typing（MST）. Methods Based on the genomes of the 4 published Anaplasma strains，4 genomic senquences were analyzed by Mauve 2.3.1 software and varable spacer sequences were selected for designing primers with the bio-software Primer Premier 5.0. A total of 11 Chinese A. phagocytophilum isolates，obtained from different areas of China during 2009-2012 were assayed by the MST. Twenty two intergenic sequences for each isolate tested and the reference A. phagocytophilum strain Webster and A. phagocytophilum strain HZ were concatenated in the order of HGA-mst 1F/1R-mst 2F/2R，HGA-mst 22F/22R. Results Twenty two pairs of primers were successfully used for typing the Human granulocytic anaplasmosis（HGA） strains in the study. Those 22 intergenic sequences exhibited a great diversity among the strains tested and each of the strain tested was identified as unique genotype，according to the alignment analysis of the 22 concatenated intergenic sequences. Of these single nucleotide polymorphism（SNPs）identified in the study，the nucleotide transitions shared the highest percentage（60.2%，251/417）and then the nucleotide transversion，accounted for 23.0%（96/417）and the indel events（insertion/deletion）were observed of 16.7%（70/417）SNPs. Phylogenetic analysis indicated that the 5 strains from patients（LZ-H1，LZ-H2，LZ-H3，LZ-H4，LZ-H5）from Laizhou areas，Shandong province and 1 tick strain（LZ-T1）from Haemaphysalis longicornis collected from the same areas where the patients lived were grouped in the same clan with the reference A. phagocytophilum strain Webster and strain HZ. Beijing isolates（BJ-H1）grouped with Xinjiang isolates（XJ-H1 and XJ-H3）while another tick isolates from Laizhou areas（LZ-T2）and another Xinjiang human isolate（XJ-H2）were in the same clan，which was closely related to the isolates from severe patients in Laizhou. Conclusion Chinese HGA isolates exhibited a great diversity of intergenic regions. MST seemed a valuable tool for the detection and tracing for any endemic strains of Anaplasma during the outbreak investigations in the public health events.

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