上海交通大学学报（医学版）

›› 2012, Vol. 32 ›› Issue (7): 886-.doi: 10.3969/j.issn.1674-8115.2012.07.014

• 论著（基础研究） • 上一篇    下一篇

REP-PCR技术在光滑假丝酵母菌基因分型中的应用价值

张 炜, 郑 冰, 应春妹, 汪雅萍, 张灏旻, 杨 俊   

  1. 上海交通大学 医学院附属仁济医院检验科, 上海 200127
  • 出版日期:2012-07-28 发布日期:2012-08-17
  • 通讯作者: 应春妹, 电子信箱: ycmzh@yahoo.com.cn。
  • 作者简介:张 炜（1981—）, 男, 技师, 学士；电子信箱: 308503526@qq.com。

Application of REP-PCR in genotyping of Candida glabrata

ZHANG Wei, ZHENG Bing, YING Chun-mei, WANG Ya-ping, ZHANG Hao-min, YANG Jun   

  1. Clinical Laboratory, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200127, China
  • Online:2012-07-28 Published:2012-08-17

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摘要：

目的 评估重复序列PCR（REP-PCR）技术在光滑假丝酵母菌基因分型中的作用。方法 收集深部真菌感染患者体内分离的光滑假丝酵母菌34株，以API 20C AUX酵母菌鉴定板条鉴定菌种及生化表型。采用REP-PCR对34株光滑假丝酵母菌进行基因分型：提取真菌基因组DNA，以Care-2重复元件设计引物，PCR扩增产物电泳后运用NTSYS软件进行聚类分析，聚类树状图相似系数（SI）≥97%且无明显条带差异定为同一基因型，SI≥97%且仅相差一条条带定为亚型。比较REP-PCR分型与多位点序列分型（MLST）的辨别力指数（DP），分析具有不同生化表型光滑假丝酵母菌的REP-PCR基因分型情况。结果 REP-PCR基因分型结果显示：34株光滑假丝酵母菌分为17个基因型，其中A型8株，B型6株，C、D型各3株，E型2株，F~Q型各1株，未发现亚型。REP-PCR和MLST基因分型的DP(95%CI）分别为0.911（0.770~0.980）和0.369（0.220~0.560），两者比较差异有统计学意义（χ2=21.68, P<0.01）。34株光滑假丝酵母菌有两种生化表型，生化表型代码分别为2000040(32株)和6000040（2株），生化表型代码为60000402的2株经REP-PCR分型结果分别为D型和K型（SI=0.43）。结论 REP-PCR对光滑假丝酵母菌基因分型的辨别力较强且操作简便，适用于临床实验室对光滑假丝酵母菌的流行病学研究。

关键词: 光滑假丝酵母菌, 重复序列PCR, 多位点序列分析, 生化表型

Abstract:

Objective To investigate the role of repetitive extragenic palindromic-PCR (REP-PCR) in the genotyping of Candida glabrata. Methods Thirty-four Candida glabrata isolates from patients with deep fungal infection were collected, and the biochemical phenotypes of isolates were identified by API 20C AUX yeast identification strip. Genotyping of 34 Candida glabrata isolates was performed with REP-PCR. Fungal genome DNA was extracted, primers for PCR was designed from Care-2 repetitive elements, the products of PCR were analysed by electrophoresis, and cluster analysis was conducted with NTSYS software. Isolates with the same genotype had a similarity index (SI) ≥97% and no significant band difference, and those with SI ≥97% and one band difference were characterized as subtypes. Indexes of discriminatory power (DP) were compared between REP-PCR genotyping and multilocus sequence typing (MLST), and the REP-PCR genotyping of Candida glabrata with different biochemical phenotypes was analysed. Results REP-PCR genotyping revealed there were 17 genotypes for these 34 Candida glabrata isolates, including type A (8 isolates), type B (6 isolates), type C and D (3 isolates for each type), type E (2 isolates) and type F to Q (1 isolate for each type), and there was no subtype. DP (95% CI) of genotyping by REP-PCR and MLST were 0.911(0.770-0.980) and 0.369 (0.220-0.560) respectively, and there were significant differences between them (χ2=21.68, P<0.01). There were two biochemical phenotype codes for these 34 Candida glabrata isolates, which were 2000040 (32 isolates) and 6000040 (2 isolates), and the latter two isolates were classified into type D and type K in REP-PCR genotyping (SI=0.43). Conclusion REP-PCR is simple to operate and is powerful in genotyping of Candida glabrata, which is suitable for the epidemiological study of Candida glabrata in clinical laboratories.

Key words: Candida glabrata, repetitive extragenic palindromic-PCR, multilocus sequence typing, biochemical phenotypes