收稿日期: 2019-12-02
网络出版日期: 2020-05-11
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
Received date: 2019-12-02
Online published: 2020-05-11
Supported by
Supported by the National Science and Technology Major Program of China(2018ZX10734-404);the National Natural Science Foundation of China(81472923)
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。
刘建秀 , 高春花 , 杨玥涛 , 郑彬 , 汪俊云 . 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[J]. 中国寄生虫学与寄生虫病杂志, 2020 , 38(2) : 181 -187 . DOI: 10.12140/j.issn.1000-7423.2020.02.008
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
| [1] | Chen JP, Yang BB, Xu JN , et al. Epidemic status of kala-azar in China[ C]//Zoological Society Symposium in Sichuan Province. Chengdu, 2011: 138. (in Chinese) |
| [1] | ( 陈建平, 杨斌斌, 徐佳楠 , 等. 我国黑热病流行现状[C]// 四川省动物学会第九次会员代表大会暨第十届学术研讨会论文集. 成都, 2011: 138.) |
| [2] | Wang JY, Gao CH, Yang YT , et al. An outbreak of the desert sub-type of zoonotic visceral leishmaniasis in Jiashi, Xinjiang Uygur Autonomous Region, People’s Republic of China[J]. Parasitol Int, 2010,59(3):331-337. |
| [3] | Zhou XN, Lv S, Yang GJ , et al. Spatial epidemiology in zoonotic parasitic diseases: insights gained at the 1st International Symposium on Geospatial Health in Lijiang, China, 2007[J]. Parasit Vectors, 2009,2:10. |
| [4] | Guan LR, Qu JQ, Chai JJ . Leishmaniasis in China-present status of prevalence and some suggestions on its control[J]. Endem Dis Bull China, 2000,15(3):49-52. (in Chinese) |
| [4] | ( 管立人, 瞿靖琦, 柴君杰 . 中国利什曼病的现状和对开展防治工作的若干建议[J]. 地方病通报, 2000,15(3):49-52.) |
| [5] | Wang JY, Cui G, Chen HT , et al. Current epidemiological profile and features of visceral leishmaniasis in People’s Republic of China[J]. Parasit Vectors, 2012,5:31. |
| [6] | Guan LR, Yang YQ, Qu JQ , et al. Discovery and study of cutaneous leishmaniasis in Karamay of Xinjiang, West China[J]. Infect Dis Poverty, 2013,2(1):20. |
| [7] | Alce TM, Gokool S ,McGhie D , et al. Expression of hydrophilic surface proteins in infective stages of Leishmania donovani[J]. Mol Biochem Parasitol, 1999,102(1):191-196. |
| [8] | Haralambous C, Antoniou M, Pratlong F , et al. Development of a molecular assay specific for the Leishmania donovani complex that discriminates L. donovani/Leishmania infantum zymodemes: a useful tool for typing MON-1[J]. Diagn Microbiol Infect Dis, 2008,60(1):33-42. |
| [9] | Kimura M . A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences[J]. J Mol Evol, 1980,16(2):111-120. |
| [10] | Saitou N, Nei M . The neighbor-joining method: a new method for reconstructing phylogenetic trees[J]. Mol Biol Evol, 1987,4(4):406-425. |
| [11] | Felsenstein J . Confidence limits on phylogenies: an approach using the bootstrap[J]. Evolution, 1985,39(4):783-791. |
| [12] | Sch?nian G, Cupolillo E, Mauricio I . Molecular evolution and phylogeny of Leishmania[M] //Drug Resistance in Leishmania Parasites. Vienna: Springer Vienna, 2012: 15-44. |
| [13] | Guan LR, Yang YQ, Zhai JQ , et al. Study of cutaneous leishmaniasis in Karamay, Xinjiang[J]. Chin J Parasitol Parasit Dis, 1997,15(3):181-185. (in Chinese) |
| [13] | ( 管立人, 杨元清, 翟靖琦 . 新疆克拉玛依地区皮肤利什曼病的研究[J]. 中国寄生虫学与寄生虫病杂志, 1997,15(3):181-185.) |
| [14] | Cheng FZ, Gao CH, Yang YT , et al. Polymorphism analysis of internal transcribed spacer 1 of Leishmania isolates from different endemic areas in China[J]. Chin J Parasitol Parasit Dis, 2019,37(2):196-201. (in Chinese) |
| [14] | ( 程芳洲, 高春花, 杨玥涛 , 等. 我国不同利什曼病流行区利什曼原虫分离株核糖体内转录间隔区1的多态性分析[J]. 中国寄生虫学与寄生虫病杂志, 2019,37(2):196-201.) |
| [15] | Bu LY, Hu XS, Jing BQ, Yi TL . Sequence analysis of SSU rDNA variable regions of Leishmania isolates from hillly foci and plain foci of China[J]. Chin J Parasitol Parasit Dis, 2000,18(6):321-324. (in Chinese) |
| [15] | ( 卜玲毅, 胡孝素, 敬保迁 , 等. 我国内脏利什曼病山丘疫区与平原疫区利什曼原虫SSUrDNA多变区序列分析[J]. 中国寄生虫学与寄生虫病杂志, 2000,18(6):321-324.) |
| [16] | Bu LY, Hu XS, Jing BQ , et al. Sequence analysis of SSU rDNA variable region of different Leishmania isolates of China[J]. Pract Parasit Dis, 2001,9(1):1-3. (in Chinese) |
| [16] | ( 卜玲毅, 胡孝素, 敬保迁 , 等. 利什曼原虫中国分离株SSU rDNA多变区序列分析[J]. 实用寄生虫病杂志, 2001,9(1):1-3.) |
| [17] | Yuan DM, Qin HX, Zhang JG , et al. Phylogenetic analysis of HSP70 and cyt b gene sequences for Chinese Leishmania isolates and ultrastructural characteristics of Chinese Leishmania sp[J]. Parasitol Res, 2017,116(2):693-702. |
| [18] | Cao DP, Guo XG, Chen DL , et al. Species delimitation and phylogenetic relationships of Chinese Leishmania isolates reexamined using kinetoplast cytochrome oxidase Ⅱ gene sequences[J]. Parasitol Res, 2011,109(1):163-173. |
| [19] | Guan W, Cao DP, Sun K , et al. Phylogenic analysis of Chinese Leishmania isolates based on small subunit ribosomal RNA (SSU rRNA) and 7 spliced leader RNA (7SL RNA)[J]. Acta parasitologica, 2012,57(2):101-113. |
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