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

doi:10.16150/j.1671-2870.2019.01.015

摘要/Abstract

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

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

中图分类号:

R372

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.

WANG Guoqing, LI Zhen, PENG Yibing. Application of microsatellite polymorphism and repetitive sequence-based PCR in genotyping of Candida tropicalis[J]. Journal of Diagnostics Concepts & Practice, 2019, 18(1): 77-81.

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引物基因	引物序列	重复序列	片段大小（bp)	荧光标记
Ctrm1	F:3’-CAACAGTTGATAGATCAAGC-5’，R: 5’-CGAACTATCACTTTTAGGAG-3’	(AGA)22	370~454	FAM
Ctrm10	F: 3’-AGTTTTCCTGTTGCTGGTTG-5’，R: 5’-CATTGAGATTGGAAGAAGTG-3’	(ATG)52	315~370	ROX
Ctrm12	F: 3’-TGTGTGTCTATTACCTCCCA-5’，R: 5’-CTGTCAGTTGTACATCATCG-3’	(AC)39	234~263	FAM

型别	菌株数	样本编号	样本种类	Ctrm1	Ctrm10	Ctrm12
1	2 1 1 1	27、28 17 25 35	痰、咽拭子血液痰粪便	362/439 362/439 362/439 362/445	330/333 333/333 330/333 330/333	239/243 239/243 243/243 239/243
2	1	12	肛周	362/442	330/333	240/243
3	3 1	5、15、16 40	肛周、痰、粪便痰	371/371 371/371	320/333 320/333	237/239 236/239
4	1	49	血液	371/371	321/333	236/238
5	1	45	痰	371/371	317/336	236/236
6	1 2	37 20、21	咽拭子粪便、咽拭子	371/393 371/393	317/317 317/317	251/275 251/278
7	1	34	痰	371/398	314/320	239/239
8	1	10	粪便	371/398	321/333	236/238
9	1	39	血液	371/431	320/352	254/256
10	1	1	肛周	371/376	320/352	237/237
11	1	9	咽拭子	371/413	314/373	239/249
12	3	29、30、11	痰、中段尿、肛周	373/410	323/330	239/252
13	1	6	痰	407/413	320/320	237/239
14	1	26	中段尿	393/407	333/333	239/269
15	1	36	痰	357/393	333/333	239/239
16	1	38	血液	387/451	320/339	237/239
17	1	48	咽拭子	387/396	314/318	232/232
18	1	4	肛周	376/387	314/358	237/243
19	2	31、32	痰	379/464	320/320	237/239
20	1	44	血液	379/382	314/320	236/239
21	3 1	22、23、24 41	引流液、中段尿、咽拭子血液	398/401 398/401	314/330 314/330	237/245 245/245
22	2	42、43	血液、血液	398/422	320/330	237/239
23	2	19、46	导尿管尿液、痰	398/398	317/320	237/239
24	1	50	粪便	398/448	320/330	235/237
25	1	33	痰	398/451	320/330	239/239
26	1	8	肛周	387/387	314/320	237/239
27	1 1	2 3	粪便痰	451/451 451/451	320/330 320/330	237/237 236/236
28	1	47	中段尿	425/464	320/320	232/239
29	2 1	13、14 18	肛周、中段尿中段尿	425/470 425/470	320/320 320/320	237/241 236/241
30	1	7	肛周	425/442	320/320	237/239

菌株数	REP-PCR基因型别	微卫星多态性基因型别
21	A	1、2、3、4、5、6、7、8,9、10、11、15
11	B	16、19、25、27、28、29、30
3	C	12
2	D	17、18
9	E	21、22、23、24
1	F	13
3	G	14、20、26
共50	共7种基因型	共30种基因型