中国寄生虫学与寄生虫病杂志 ›› 2020, Vol. 38 ›› Issue (2): 181-187.doi: 10.12140/j.issn.1000-7423.2020.02.008

• 论著 • 上一篇    下一篇

利什曼原虫K26序列应用于我国利什曼原虫分离株鉴定的价值分析

刘建秀, 高春花, 杨玥涛, 郑彬, 汪俊云*()   

  1. 中国疾病预防控制中心寄生虫病预防控制所,国家热带病研究中心,世界卫生组织热带病合作中心,科技部国家级热带病国际联合研究中心,卫生部寄生虫病原与媒介生物学重点实验室,上海 200025
  • 收稿日期:2019-12-02 出版日期:2020-04-30 发布日期:2020-05-11
  • 通讯作者: 汪俊云
  • 作者简介:刘建秀(1988-),女,硕士研究生,从事病原生物学研究。E-mail: 784158390@qq.com
  • 基金资助:
    国家重大科技专项(2018ZX10734-404);国家自然科学基金(81472923)

Evaluation of the value of K26 sequence applied in identification of Leishmania isolates in China

Jian-xiu LIU, Chun-hua GAO, Yue-tao YANG, Bin ZHENG, Jun-yun WANG*()   

  1. National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; WHO Collaborating Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Ministry of Science and Technology; Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai 200025, China
  • Received:2019-12-02 Online:2020-04-30 Published:2020-05-11
  • Contact: Jun-yun WANG
  • Supported by:
    Supported by the National Science and Technology Major Program of China(2018ZX10734-404);the National Natural Science Foundation of China(81472923)

摘要:

目的 评价利什曼原虫K26序列应用于我国利什曼原虫分离株鉴定的价值。方法 收集我国不同流行区的利什曼原虫16个分离株和6个国际参照株,培养后收集前鞭毛体,提取DNA,扩增K26序列并测序,应用ClustalX2进行序列比对,从GenBank下载同源性较高的利什曼原虫分离株的K26序列,构建系统进化树,分析我国不同流行区的利什曼原虫分离株的亲缘关系。结果 我国16个利什曼原虫分离株和国际参照株DD8均扩增出单一条带,而5个非杜氏利什曼原虫复合体虫株均未扩增出任何条带。序列分析结果表明,我国人源型利什曼病流行区的3个分离株801、KS-2和KS-6均扩增出920 bp片段,自然疫源型利什曼病流行区的4个分离株XJ771、JIASHI-1、JIASHI-2和JIASHI-5均扩增出491 bp片段,人犬共患型利什曼病流行区4个分离株Cy、SC6、Hebei-Lv和Shanxi-Yang均扩增出404 bp片段,皮肤利什曼病流行区的5个分离株KXG-Xu、KXG-Liu、KXG-65、KXG-918和KXG-927均扩增出449 bp片段,且各流行区内虫株间的序列一致性均为100%。各流行区之间的虫株间的序列一致性较低,为40.2%~91.4%。系统进化树分析结果显示,我国16个利什曼原虫分离株聚集成2个群3个亚群,其中KXG-Xu、KXG-Liu、KXG-65、KXG-918、KXG-927、XJ771、JIASHI-1、JIASHI-2、JIASHI-5分离株聚集为A亚群,Cy、SC6、Hebei-Lv、Shanxi-Yang与婴儿利什曼原虫聚集为B亚群,A亚群和B亚群聚集为Ⅰ群;801、KS-2和KS-6分离株与杜氏利什曼原虫聚集为C亚群,进一步与国际参照株DD8聚集为Ⅱ群。结论 利什曼原虫K26序列可应用于我国利什曼原虫分离株的鉴定。

关键词: 利什曼病, 利什曼原虫, 分离株, K26, 系统进化树, 中国

Abstract:

Objective To evaluate the application of Leishmania K26 sequence in identification of Leishmania isolates in China.Methods Sixteen Leishmania isolates collected from different domestic endemic areas and 6 international reference strains were cultured for collection of promastigotes, from which DNA was extracted for amplification of K26 sequence by PCR. The K26 sequence was confirmed by sequencing. The sequence alignment was made using the ClustalX2 software. A phylogenetic tree was constructed based on the K26 sequences of highly-homologous Leishmania isolates downloaded from the GenBank database, to analyze the genetic relationship among the Leishmania isolates from different endemic areas in China.Results PCR amplification showed a specific band from all the 16 isolates and the DD8 international reference strain, while no specific band was found from five non-L. donovani complex strains. Sequence analysis indicates that all 3 isolates 801, KS-2 and KS-6 from the endemic areas of anthroponotic leishmaniasis generated a 920 bp fragment in PCR; 4 isolates XJ771, JIASHI-1, JIASHI-2 and JIASHI-5 from endemic areas of natural-sourced leishmaniasis all generated a 491 bp fragment in amplification reaction; 4 strains Cy, SC6, Hebei-Lv and Shanxi-Yang from endemic areas of zoonosis (specifically canine) leishmaniasis generated a 404 bp in amplification; 5 strains KXG-Xu, KXG-Liu, KXG-65, KXG-918 and KXG-927 from endemic areas of cutaneous leishmaniasis all generated a 449 bp fragment in amplification. Among the endemic areas, the K26 sequences of intra-area isolates showed 100% homology, while the sequences of enter-area isolates displayed a lower homology (40.2%-91.4%). Phylogenetic tree analysis showed that the 16 isolates of Leishmania in China clustered into 2 groups and 3 subgroups, among which nine isolates including KXG-Xu, KXG-Liu, KXG-65, KXG-918, KXG-927, XJ771, JIASHI-1, JIASHI-2, and JIASHI-5 clustered into subgroup A, while five isolates Cy, SC6, Hebei-Lv, Shanxi-Yang and L. infantum clustered into subgroup B, and both subgroups clustered into groupⅠ. The 801, KS-2 and KS-6 isolates clustered with L. donovani as subgroup C, which further clustered with the DD8 international reference isolate into group Ⅱ.Conclusion The K26 sequences of Leishmania can be used for identification of Leishmania isolates in China.

Key words: Leishmaniasis, Leishmania, Isolate, K26, Phylogenetic tree, China

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