Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology.
Internal transcribed spacer region (ITS) sequencing is the most extensively used technology for accurate molecular identification of fungal pathogens in clinical microbiology laboratories. Intra-genomic ITS sequence heterogeneity, which makes fungal identification based on direct sequencing of PCR products difficult, has rarely been reported in pathogenic fungi. During the process of performing ITS sequencing on 71 yeast strains isolated from various clinical specimens, direct sequencing of the PCR products showed ambiguous sequences in six of them. After cloning the PCR products into plasmids for sequencing, interpretable sequencing electropherograms could be obtained. For each of the six isolates, 10-49 clones were selected for sequencing and two to seven intra-genomic ITS copies were detected. The identities of these six isolates were confirmed to be Candida glabrata (n=2), Pichia (Candida) norvegensis (n=2), Candida tropicalis (n=1) and Saccharomyces cerevisiae (n=1). Multiple sequence alignment revealed that one to four intra-genomic ITS polymorphic sites were present in the six isolates, and all these polymorphic sites were located in the ITS1 and/or ITS2 regions. We report and describe the first evidence of intra-genomic ITS sequence heterogeneity in four different pathogenic yeasts, which occurred exclusively in the ITS1 and ITS2 spacer regions for the six isolates in this study.
Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China. zhaoying28062806@163.com.|Department of Microbiology, The University of Hong Kong, Hong Kong. zhaoying28062806@163.com.
Ying
Y
Zhao
Department of Microbiology, The University of Hong Kong, Hong Kong. microbioct@connect.hku.hk.
Chi-Ching
CC
Tsang
Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China. cjtcxiaomeng@aliyun.com.
Meng
M
Xiao
Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China. zzujingwei@163.com.|Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China. zzujingwei@163.com.
Jingwei
J
Cheng
Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China. xycpumch@139.com.
Yingchun
Y
Xu
Department of Microbiology, The University of Hong Kong, Hong Kong. skplau@hku.hk.|State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong. skplau@hku.hk.|Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong. skplau@hku.hk.|Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong. skplau@hku.hk.
Susanna K P
SK
Lau
Department of Microbiology, The University of Hong Kong, Hong Kong. pcywoo@hku.hk.|State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong. pcywoo@hku.hk.|Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong. pcywoo@hku.hk.|Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong. pcywoo@hku.hk.
Patrick C Y
PC
Woo
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D041221:Candida glabrata / Q000235:genetics; D041022:Candida tropicalis / Q000235:genetics; D002177:Candidiasis / Q000175:diagnosis* / Q000382:microbiology; D004271:DNA, Fungal / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000235:genetics*; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D008480:Mediastinitis / Q000175:diagnosis* / Q000382:microbiology; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D010538:Peritonitis / Q000175:diagnosis* / Q000382:microbiology; D010843:Pichia / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012189:Retrospective Studies; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D021903:DNA, Ribosomal Spacer
heterogeneity; internal transcribed spacer region; molecular identification; sequencing; yeast