北京地区志贺菌多位点可变数目串联重复序列分析分子分型研究

Multilocus variable-number tandem-repeat analysis for molecular subtypes of Shigeila isolates in Beijing

目的筛选适用于志贺菌不同血清群分子分型的可变数目串联重复序列（VNrFR）位点，探索建立多位点可变数目串联重复序列分析（MLVA）分型方法，应用于菌株的分子特征分析。方法从2001—2009年北京地区痢疾监测中收集到的志贺菌菌株中，根据每年监测到的菌株数量和各种血清型别按15％的比例抽取，共选择180株菌，其中宋内志贺菌50株，福氏志贺菌130株。对18个VNTR位点进行多态性筛选后，保留10个VNTR位点（sh1—sh10）构成3组多重PCR方法进行检测，利用毛细管片段分析对180株菌进行检测和MLVA分子分型。结果筛选的10个VNTR位点在180株志贺菌菌株中等位基因数目为2-11种，多态性分辨系数（D值）为0．158—0．766。在不同血清群间，10个位点的多态性不同，其中s怕在福氏志贺菌、sh2和sh3在宋内志贺菌中均只有1种等位基因。180株志贺菌分成84种MLVA型别，口值为0．967（95％CI：0．956～0．978），其中13C株福氏志贺菌，分成63种型别，命名为TF001-TF063，TF001、TF002和TF005为主要型别，分别为17、16和15株；50株宋内志贺菌，分成21个型别，命名为TS001～TS021，TS002和TS001为主要型别，分别为14、7株。结论初步建立了志贺菌10个VNTR位点MLVA分型方法。通过MLVA分析，揭示志贺菌北京分离株分子型别比较分散，存在多克隆来源。

Objective Selecting variable-number tandem-repeat （ VNTR ） loci for different serogroups of Shigella spp to explore and establish multilocus variable-number tandem-repeat analysis （MLVA） method, in order to study the molecular characteristic of the isolated strains. Methods Of the Shigella strains found by dysentery surveillance in Beijing from 2001 to 2009, 180 strains were selected for this study, according to the number and serotypes of the surveillant strains, at the ratio of 15% ; including 50 strains of Shigella sonnei and 130 strains of Shigella flexneri. After screening the polymorphism of the 18 VNTR loci, 10 VNTR loci （ sh1 - sh10） were retained and constructed three groups of muhi-PCR methods to detect all the 180 strains and analyze MLVA molecular subtypes using capillary segments. Results A rangeof 2 to 11 alleles were found on the 10 VNTR loci among the 180 Shigella strains, with a diversity index value （ D value ） between 0. 158 and 0. 766. The 10 loci showed diwersity in different serogroups, such as only one allele found in sh6 of Shigella flexneri, sh2 and sh3 of Shigella sonnei individually. The isolated 180 strains were divided into 84 MLVA subtypes, with a resolution ratio D value at 0. 967 （95% CI： 0. 956 -0. 978）. The 130 strains of Shigellaflexneri were divided into 63 subtypes, named as TF001 - TF063 ; among which TF001, TF002 and TF005 were the dominant subtypes, accounting to 17, 16 and 15 strains respectively. The 50 strains of Shigella sonnei were divided into 21 subtypes, named as TS001- TS021; among which TS002 （14 strains ） and TS001 （7 strains ） were the dominant subtypes. Conclusion MLVA subtyping method including 10 VNTR loci was preliminarily developed. The MLVA eluster analysis revealed that the subtypes of Shigella strains isolated in Beijing were diverse, and suggested the possibility of multiple-clone souree.