诊断学理论与实践

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2019 , Vol. 18 >Issue 1: 77 - 81

DOI: https://doi.org/10.16150/j.1671-2870.2019.01.015

论著

微卫星多态性和重复序列PCR分析技术在热带假丝酵母菌基因分型中的应用

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  • 1. 上海交通大学医学院附属瑞金医院检验科，上海 200025
    2. 上海中医药大学附属龙华医院检验科，上海 200032

收稿日期: 2018-06-25

  网络出版日期: 2019-02-25

基金资助

上海市黄浦区医疗卫生重点研究发展专科科研基金项目(HWZFK201801);上海市黄浦区科技项目(HKW201505)

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Application of microsatellite polymorphism and repetitive sequence-based PCR in genotyping of Candida tropicalis

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  • 1. Clinical Laboratory, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
    2. Clinical Laboratory, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China

Received date: 2018-06-25

  Online published: 2019-02-25

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摘要

目的 评估微卫星多态性分析技术和重复序列PCR分析技术在临床热带假丝酵母菌基因分型中的应用。方法 收集2014年8月到2015年11月来自上海4家医院临床分离的热带假丝酵母菌共50株，分别以Ca21、Ca22、Com21两两组合为引物，选用最合适的引物组合进行重复序列聚合酶链反应（repetitive extragenic palindromic polymerase chain reaction，REP-PCR）后电泳获得REP-PCR分型。采用Ctrm1、Ctrm10、Ctrm12这3种微卫星标记进行微卫星多态性分析。比较2种基因分型方法的结果和分辨力。结果 REP-PCR以Ca21-Com21引物组合的分型效果最好，50株热带假丝酵母菌经REP-PCR分型得到共7种REP-PCR型，鉴别力指数（index of discriminatory power, DP）为0.75。经多态性分型得30种基因型，DP为0.97。结论 微卫星多态性分析简便、快捷，分辨力高于REP-PCR检测，可作为实验室基因分型的首选方法。

关键词： 热带假丝酵母菌; 基因分型; 重复序列PCR; 微卫星多态性技术

本文引用格式

汪国庆, 李贞, 彭奕冰 . 微卫星多态性和重复序列PCR分析技术在热带假丝酵母菌基因分型中的应用[J]. 诊断学理论与实践, 2019 , 18(1) : 77 -81 . DOI: 10.16150/j.1671-2870.2019.01.015

Abstract

Objective: To evaluate the application of microsatellite polymorphism in genotyping of Candida tropicalis. Methods: From August 2014 to November 2015, 50 clinical isolates of Candida tropicalis were collected form 4 hospitals, including Ruijin Hospital, Children’s Hospital, the First People’s Hospital and Zhongshan Hospital. Primers Ca21, Ca22, Com21 were used in pairs to find the best suitable pairs for the repetitive extragenic palindromic polymerase chain reaction (REP-PCR) genotyping. Three microsatellite markers Ctrm1, Ctrom10, Ctrm12 were used to analyze the microsatellite polymorphism. Finally, the discriminatory results of the two genotyping methods were compared. Result: The combination of Ca21-Com21 primers had the best effect in REP-PCR genotyping. Seven REP-PCR types A-G were found in 50 isolates of Candida tropicalis. The index of discriminatory power was 0.75. The 50 isolates were classified into 30 genotypes by the microsatellites polymorphism. The index of discriminatory power was 0.97. Conclusions: Microsatellite polymorphism is a simple and rapid method for molecular typing with higher discrimination power than REP-PCR. Therefore, microstellite polymorphism is the preferred choice in clinical laboratories.

Key words： Candida tropicalis; Genotype; Repetitive extragenic palindromicpolymerase chain reaction; Microsatellite polymorphism

参考文献

[1] Chandra J, Mukherjee PK. Candida Biofilms: Development, Architecture, and Resistance[J/OL]. Microbiol Spectr, 2015-08[2018-07-17]https://www.ncbi.nlm.nih.gov/pubmed/26350306.
[2] Safavieh M, Coarsey C, Esiobu N, et al. Advances in Candida detection platforms for clinical and point-of-care applications[J]. Crit Rev Biotechnol, 2017, 37(4):441-458.
[3] Chin VK, Lee TY, Rusliza B, et al. Dissecting Candida albicans Infection from the Perspective of C. albicans Virulence and Omics Approaches on Host-Pathogen Interaction: A Review[J]. Int J Mol Sci, 2016, 17(10)pii:E1643.
[4] Arrua JM, Rodrigues LA, Pereira FO, et al. Prevalence of Candida tropicalis and Candida krusei in onychomycosis in João Pessoa, Paraiba, Brazil from 1999 to 2010[J]. An Acad Bras Cienc, 2015, 87(3):1819-1822.
[5] Fernandes JA, Prandini TH, Castro MD, et al. Evolution and Application of Inteins in Candida species: A Review[J]. Front Microbiol, 2016, 7:1585.
[6] Wang H, Xu J, Guo H, et al. Patterns of human oral yeast species distribution on Hainan Island in China[J]. Mycopathologia, 2013, 176(5-6):359-368.
[7] Wang H, Xiao M, Chen SC, et al. In vitro susceptibilities of yeast species to fluconazole and voriconazole as determined by the 2010 National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) study[J]. J Clin Microbiol, 2012, 50(12):3952-3959.
[8] Parente-Rocha JA, Bailão AM, Amaral AC, et al. Antifungal resistance, metabolic routes as drug targets, and new antifungal agents: An overview about endemic dimorphic Fungi[J]. Mediators Inflamm, 2017, 2017:9870679.
[9] Yang S, Liao Y, Cong L, et al. In vitro interactions between non-steroidal anti-inflammatory drugs and antifungal agents against planktonic and biofilm forms of trichosporon asahii[J]. PLoS One, 2016, 11(6):e0157047.
[10] Pitout JD, Campbell L, Church DL, et al. Using a commercial DiversiLab semiautomated repetitive sequence-based PCR typing technique for identification of Escherichia coli clone ST131 producing CTX-M-15[J]. J Clin Microbiol, 2009, 47(4):1212-1215.
[11] 江芩, 董丹凤, 俞焙秦, 等. 重复序列PCR与多位点分型技术在热带假丝酵母菌基因分型中的比较[J]. 检验医学, 2012, 27(11):1673-8640.
[12] Higgins PG, Hujer AM, Hujer KM, et al. Interlaboratory reproducibility of DiversiLab rep-PCR typing and clustering of Acinetobacter baumannii isolates[J]. J Med Microbiol, 2012, 61(Pt 1):137-141.
[13] Redkar RJ, Dubé MP, McCleskey FK, et al. DNA fingerprinting of Candida rugosa via repetitive sequence-based PCR[J]. J Clin Microbiol, 1996, 34(7):1677-1681.
[14] Wu Y, Zhou HJ, Che J, et al. Multilocus microsatellite markers for molecular typing of Candida tropicalis isolates[J]. BMC Microbiol, 2014, 14:245.
[15] Hunter PR, Gaston MA. Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity[J]. J Clin Microbiol, 1988, 26(11):2465-2466.
[16] Dacal E, Saugar JM, de Lucio A, et al. Prevalence and molecular characterization of Strongyloides stercoralis, Giardia duodenalis, Cryptosporidium spp, and Blastocystis spp. isolates in school children in Cubal, Western Angola[J]. Parasit Vectors, 2018, 11(1):67.
[17] Wu Y, Zhou HJ, Che J, et al. Multilocus microsatellite markers for molecular typing of Candida tropicalis isolates[J]. BMC Microbiol, 2014, 14:245.
[18] Chou HH, Lo HJ, Chen KW, et al. Multilocus sequence typing of Candida tropicalis shows clonal cluster enriched in isolates with resistance or trailing growth of fluconazole[J]. Diagn Microbiol Infect Dis, 2007, 58(4):427-433.
[19] Blanchard AM, Jolley KA, Maiden MCJ, et al. The Applied Development of a Tiered Multilocus Sequence Ty-ping (MLST) Scheme for Dichelobacter nodosus[J]. Front Microbiol, 2018, 9:551.
[20] Redkar RJ, Dubé MP, McCleskey FK, et al. DNA fingerprinting of Candida rugosa via repetitive sequence-based PCR[J]. J Clin Microbiol, 1996, 34(7):1677-1681.
[21] Bagshaw ATM. Functional Mechanisms of Microsatellite DNA in Eukaryotic Genomes[J]. Genome Biol Evol, 2017, 9(9):2428-2443.
[22] Amouri I, Sellami H, Abbes S, et al. Microsatellite analysis of Candida isolates from recurrent vulvovaginal candidiasis[J]. J Med Microbiol, 2012, 61(Pt 8):1091-1096.
[23] Fan X, Xiao M, Liu P, et al. Novel Polymorphic Multilocus Microsatellite Markers to Distinguish Candida tropicalis Isolates[J]. PLoS One, 2016, 11(11):e0166156.
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