title: hsp65 PCR-restriction analysis (PRA) with capillary electrophoresis in comparison to three other methods for identification of Mycobacterium species.
abstract: We developed a scheme for rapid identification of Mycobacterium species using an automated fluorescence capillary electrophoresis instrument. A 441-bp region of the hsp65 gene was examined using PCR-restriction analysis (PRA). The assay was initially evaluated on 38 reference strains. The observed sizes of restriction fragments were consistently smaller than the real sizes for each of the species as deduced from the sequence analysis (mean variance=7bp). Nevertheless, the obtained PRA patterns were highly reproducible and resulted in correct species identifications. A blind test was then successfully performed on 64 test isolates previously characterized by conventional biochemical methods, a commercial INNO-LiPA Mycobacteria assay and/or sequence determination of the 5' end of 16S rRNA gene. A total of 14 of 64 isolates were erroneously identified by conventional methods (78% accuracy). In contrast, PRA performed very well in comparison with the LiPA (89% concordance) and especially with DNA sequencing (93.3% of concordant results). Also, PRA identified seven isolates representing five previously unreported hsp65 alleles. We conclude that hsp65 PRA based on automated capillary electrophoresis is a rapid, simple and reliable method for identification of mycobacteria.
authors.affiliation: Mycobacteriology Unit, Department of Microbiology, Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium.
authors.forename: Anna
authors.initials: A
authors.lastname: Sajduda
authors.forename: Anandi
authors.initials: A
authors.lastname: Martin
authors.forename: Françoise
authors.initials: F
authors.lastname: Portaels
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Palomino
mesh_terms: D000818:Animals; D001331:Automation; D001426:Bacterial Proteins / Q000235:genetics*; D001431:Bacteriological Techniques / Q000379:methods*; D018834:Chaperonin 60 / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004783:Environmental Microbiology; D006801:Humans; D009161:Mycobacterium / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D009164:Mycobacterium Infections / Q000382:microbiology / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; C074119:heat-shock protein 65, Mycobacterium