黄振洲, 于可艺, 代航, 蔡红艳, 徐潇, 王多春. 麦氏弧菌多位点序列分型方法的建立与应用[J]. 疾病监测, 2020, 35(1): 75-80. DOI: 10.3784/j.issn.1003-9961.2020.01.017
引用本文: 黄振洲, 于可艺, 代航, 蔡红艳, 徐潇, 王多春. 麦氏弧菌多位点序列分型方法的建立与应用[J]. 疾病监测, 2020, 35(1): 75-80. DOI: 10.3784/j.issn.1003-9961.2020.01.017
Zhenzhou Huang, Keyi Yu, Hang Dai, Hongyan Cai, Xiao Xu, Duochun Wang. Establishment and application of multilocus sequence typing for Vibrio metschnikovii[J]. Disease Surveillance, 2020, 35(1): 75-80. DOI: 10.3784/j.issn.1003-9961.2020.01.017
Citation: Zhenzhou Huang, Keyi Yu, Hang Dai, Hongyan Cai, Xiao Xu, Duochun Wang. Establishment and application of multilocus sequence typing for Vibrio metschnikovii[J]. Disease Surveillance, 2020, 35(1): 75-80. DOI: 10.3784/j.issn.1003-9961.2020.01.017

麦氏弧菌多位点序列分型方法的建立与应用

Establishment and application of multilocus sequence typing for Vibrio metschnikovii

  • 摘要:
    目的建立针对麦氏弧菌的多位点序列分型(MLST)方法,评价该方法的分型效果,并对收集的麦氏弧菌进行MLST分析。
    方法筛选出7个管家基因(ftsZgapAmreBgyrBpurMrecAropA),设计特异引物,在17株麦氏弧菌中PCR扩增管家基因序列。 用DnaSP 6.12.03软件和Split tree 4.0软件评价基因多态性、中性进化和基因重组情况;根据7个管家基因的序列差异获得对应的序列分型(ST)型别,用BioNumerics 7.1软件对不同ST型别构建最小生成树和聚类图,并用eBURST 3.0软件计算克隆复合体(CCs)。
    结果所选的管家基因具有足够多的等位基因位点、序列差异和足够高的分辨率;7个管家基因的Split tree对所有麦氏弧菌的聚类一致;中性试验结果显示,7个管家基因在进化过程中受遗传漂变的影响较小。 MLST将17株麦氏弧菌分成14个ST型别,大多数菌株(64.70%)均单独形成一个ST型别,其中ST007形成了可能的优势克隆群。 这些菌株之间存在相对较远的遗传距离,在垂直传播上呈现出潜在的地域聚集性。
    结论本研究建立的麦氏弧菌的MLST方法能分析麦氏弧菌的种群结构和遗传进化关系。

     

    Abstract:
    ObjectiveTo establish a multilocus sequence typing (MLST) assay for Vibrio metschnikovii, evaluate the molecular typing effect of the MLST assay, and perform MLST analysis on the collected V. metschnikovii strains.
    MethodsSeven housekeeping genes (ftsZ, gapA, mreB, gyrB, purM, recA, ropA) sequences in 17 V. metschnikovii strains were amplified with PCR by using self-designed specific primers. The gene polymorphism, neutral evolution and gene recombination were evaluated by using software DnaSP 6.12.03 and software Split tree 4.0, and corresponding sequence types (ST) were obtained based on the sequence differences among 7 housekeeping genes. The minimum spanning tree and clustering map were constructed for different ST types by using software BioNumerics 7.1, and clonal complexes (CCs) were calculated by using software eBURST 3.0.
    ResultsThe selected 7 housekeeping genes were suitable for MLST with sufficient allele loci, sequence differences and high resolving power. The split tree clustering of 7 housekeeping genes was consistent with all of V. metschnikovii strains, and the neutrality tests showed that 7 housekeeping genes were less subject to genetic drift during evolution. MLST divided 17 V. metschnikovii strains into 14 STs, and most of them (64.70 %) formed a single ST respectively, of which ST 007 was probably a potential predominant clonal complex. There was a relatively long genetic distance between these strains, and may have specific geographical distribution in vertical transmission.
    ConclusionThe MLST assay established in this study has great capability in analyzing the population structure and evolution relationship of V. metschnikovii.

     

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