Identification of Mycobacterium species by multiple-fluorescence PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene

Citation
Lm. Gillman et al., Identification of Mycobacterium species by multiple-fluorescence PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene, J CLIN MICR, 39(9), 2001, pp. 3085-3091
Citations number
25
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Clinical Immunolgy & Infectious Disease",Microbiology
Journal title
JOURNAL OF CLINICAL MICROBIOLOGY
ISSN journal
0095-1137 → ACNP
Volume
39
Issue
9
Year of publication
2001
Pages
3085 - 3091
Database
ISI
SICI code
0095-1137(200109)39:9<3085:IOMSBM>2.0.ZU;2-Q
Abstract
Identification of mycobacteria to the species level by growth-based methodo logies is a process that has been fraught with difficulties due to the long generation times of mycobacteria. There is an increasing incidence of unus ual nontuberculous mycobacterial infections, especially in patients with co ncomitant immunocompromised states, which has led to the discovery of new m ycobacterial species and the recognition of the pathogenicity of organisms that were once considered nonpathogens. Therefore, there is a need for rapi d and sensitive techniques that can accurately identify all mycobacterial s pecies. Multiple-fluorescence-based PCR and subsequent single-strand confor mation polymorphism (SSCP) analysis (MF-PCR-SSCP) of four variable regions of the 16S rRNA gene were used to identify species-specific patterns for 30 of the most common mycobacterial human pathogens and environmental isolate s. The species-specific SSCP patterns generated were then entered into a da tabase by using BioNumerics, version 1.5, software with a pattern-recogniti on capability, among its multiple uses. Patient specimens previously identi fied by 16S rRNA gene sequencing were subsequently tested by this method an d were identified by comparing their patterns with those in the reference d atabase. Fourteen species whose SSCP patterns were included in the database were correctly identified. Five other test organisms were correctly identi fied as unique species or were identified by their closest relative, as the y were not in the database. We propose that MF-PCR-SSCP offers a rapid, spe cific, and relatively inexpensive identification tool for the differentiati on of mycobacterial species.