Identification of Mycobacterium species by PCR-restriction fragment lengthpolymorphism analyses using fluorescence capillary electrophoresis

We developed a scheme for the rapid identification of Mycobacterium species based upon PCR amplification of polymorphic genetic regions with fluoresce nt primers followed by restriction and analysis by fluorescence capillary e lectrophoresis. Mycobacterium species were identified by restriction enzyme analysis of a 439-bp segment of the 65-kDa heat shock protein gene (labele d [both strands] at the 5' end with 4,7,2',7'-tetrachloro-6-carboxyfluoresc ein) using Haem and BstEII and of a 475-bp hypervariable region of the 16S rRNA gene (labeled [both strands] at the 5' end with 6-carboxyfluorescein) using HaeIII and CfoI. Samples were analyzed on an automated fluorescence c apillary electrophoresis instrument, and labeled fragments were sized by co mparison with an internal standard. DNA templates were prepared with pure c ultures of type strains. In all, we analyzed 180 strains, representing 22 M ycobacterium species, and obtained distinctive restriction fragment length polymorphism (RFLP) patterns for 19 species, Three members of the Mycobacte rium tuberculosis complex had a common RFLP pattern. A computerized algorit hm which eliminates subjectivity from pattern interpretation and which is c apable of identifying the species within a sample was developed. The conven ience and short preparatory time of this assay make it comparable to conven tional methodologies such as highperformance liquid chromatography and hybr idization assays for identification of mycobacteria.