MLVA方法在军团菌分子分型中的应用评价

Assessment on the application of multiple-locus variable-number tandem repeat analysis forLegionella subtyping

目的探讨MLVA方法在军团菌分型中的可应用性。方法选用文献报道的9个军团菌VNTR位点,对我国环境水中分离的81株Lp1型军团菌菌株和2株参考菌株进行分子分型研究,并与PFGE和SBT分型结果进行了比较。结果 81株分离菌株9个VNTR位点的等位基因型别数分别为6,3,4,2,3,3,2,4和7,其中部分菌株的某些位点无扩增产物或产物为非目的片断。7个位点PCR产物具有重复单元;Lpms3位点PCR产物无重复单元;Lpms31位点部分菌株PCR产物有重复单元,另一些菌株无重复单元。对于具有重复单元的序列,不同菌株间以及同一菌株内各重复单元的序列变异均较大,3个位点存在不完整的重复单元。根据8个位点的PCR产物电泳结果,81株Lp1型分离菌株分为19个MLVA型。通过PFGE和SBT方法,81株Lp1型分离菌株分别分为46种PFGE带型和21种ST型。结论 MLVA方法的分型能力不及PFGE方法,而与SBT方法分型能力相当。但是,MLVA方法操作简单、成本低廉,而且可对培养阴性的标本进行分型,因此,该方法适合在基层推广应用。

To evaluate the applicability of multiple-locus variable-number tandem repeat analysis for Legionella subtyping,9 VNTR loci were selected to type 81 Legionella pneumophila serogroup 1 strains isolated from environmental water in China.There were 6,3,4,2,3,3,2,4 and 7 different products amplified from 9 VNTR loci respectively.No specific amplification was obtained from several loci of some strains.Repeated sequences were identified in PCR products of 7 loci.No repeated sequence was found in all sequenced products from locus Lpms3 and in some products from locus Lpms31.The sequences of tandem repeats varied within the same isolate as among isolates.Incomplete tandem repeats were found in 3 loci.Based on the polymorphisms of 8 loci,81 Lp1 strains could be classified into 19 types.Comparatively,these strains could be divided into 46 species and 21 molecular types by PFGE and SBT respectively.So,MLVA was less sensitive for Legionella subtyping.But it was easy and low-cost,and could be used in relatively simply equipped laboratories.