title: 16S-23S Internal Transcribed Spacer Region PCR and Sequencer-Based Capillary Gel Electrophoresis has Potential as an Alternative to High Performance Liquid Chromatography for Identification of Slowly Growing Nontuberculous Mycobacteria.
abstract: Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 species) of SG-NTM were included. Identification was compared with that achieved by high performance liquid chromatography (HPLC), in-house PCR and 16S/ITS sequencing. Isolates of all species yielded a SCGE profile comprising a single fragment length (or peak) except for M. scrofulaceum (two peaks). SCGE peaks of ATCC strains were distinct except for peak overlap between Mycobacterium kansasii and M. marinum. Of clinical/environmental strains, unique peaks were seen for 7/17 (41%) species (M. haemophilum, M. kubicae, M. lentiflavum, M. terrae, M. kansasii, M. asiaticum and M. triplex); 3/17 (18%) species were identified by HPLC. There were five SCGE fragment length types (I-V) each of M. avium, M. intracellulare and M. gordonae. Overlap of fragment lengths was seen between M. marinum and M. ulcerans; for M. gordonae SCGE type III and M. paragordonae; M. avium SCGE types III and IV, and M. intracellulare SCGE type I; M. chimaera, M. parascrofulaceum and M. intracellulare SCGE types III and IV; M. branderi and M. avium type V; and M. vulneris and M. intracellulare type V. The ITS-SCGE method was able to provide the first line rapid and reproducible species identification/screening of SG-NTM and was more discriminatory than HPLC.
authors.affiliation: Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR - Pathology West, Westmead Hospital, Westmead, NSW, Australia.
authors.forename: Shradha
authors.initials: S
authors.lastname: Subedi
authors.affiliation: Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR - Pathology West, Westmead Hospital, Westmead, NSW, Australia.
authors.forename: Fanrong
authors.initials: F
authors.lastname: Kong
authors.affiliation: Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR - Pathology West, Westmead Hospital, Westmead, NSW, Australia.|Centre for Infectious Diseases and Microbiology - Public Health, Westmead Hospital, Westmead, New South Wales, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Jelfs
authors.affiliation: Department of Infectious Diseases and Microbiology, Concord Hospital, Concord, New South Wales, Australia.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Gray
authors.affiliation: Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Xiao
authors.affiliation: Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR - Pathology West, Westmead Hospital, Westmead, NSW, Australia.|Centre for Infectious Diseases and Microbiology - Public Health, Westmead Hospital, Westmead, New South Wales, Australia.|Centre for Infectious Diseases and Microbiology, Westmead Hospital and the Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, Sydney, NSW, Australia.
authors.forename: Vitali
authors.initials: V
authors.lastname: Sintchenko
authors.affiliation: Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR - Pathology West, Westmead Hospital, Westmead, NSW, Australia.|Centre for Infectious Diseases and Microbiology - Public Health, Westmead Hospital, Westmead, New South Wales, Australia.|Centre for Infectious Diseases and Microbiology, Westmead Hospital and the Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, Sydney, NSW, Australia.
authors.forename: Sharon C-A
authors.initials: SC
authors.lastname: Chen
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D021903:DNA, Ribosomal Spacer / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D019075:Electrophoresis, Capillary; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D009165:Mycobacterium Infections, Nontuberculous / Q000382:microbiology / Q000473:pathology; D009170:Nontuberculous Mycobacteria / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D013045:Species Specificity
chemical_list: D021903:DNA, Ribosomal Spacer; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S