Identification of Mycobacterium species by PCR-restriction fragment length polymorphism 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 fluorescent primers followed by restriction and analysis by fluorescence capillary electrophoresis. Mycobacterium species were identified by restriction enzyme analysis of a 439-bp segment of the 65-kDa heat shock protein gene (labeled [both strands] at the 5' end with 4,7,2',7'-tetrachloro-6-carboxyfluorescein) using HaeIII 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 capillary electrophoresis instrument, and labeled fragments were sized by comparison with an internal standard. DNA templates were prepared with pure cultures of type strains. In all, we analyzed 180 strains, representing 22 Mycobacterium species, and obtained distinctive restriction fragment length polymorphism (RFLP) patterns for 19 species. Three members of the Mycobacterium tuberculosis complex had a common RFLP pattern. A computerized algorithm which eliminates subjectivity from pattern interpretation and which is capable of identifying the species within a sample was developed. The convenience and short preparatory time of this assay make it comparable to conventional methodologies such as high-performance liquid chromatography and hybridization assays for identification of mycobacteria.
Hernandez
S M
SM
Division of AIDS, Sexually Transmitted Diseases, and Tuberculosis Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.
Morlock
G P
GP
Butler
W R
WR
Crawford
J T
JT
Cooksey
R C
RC
D001426:Bacterial Proteins / Q000235:genetics; D015373:Bacterial Typing Techniques; D001483:Base Sequence; D018834:Chaperonin 60; D018833:Chaperonins / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D005069:Evaluation Studies as Topic; D006801:Humans; D009161:Mycobacterium / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D009164:Mycobacterium Infections / Q000175:diagnosis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D014376:Tuberculosis / Q000175:diagnosis
D001426:Bacterial Proteins; D018834:Chaperonin 60; D017931:DNA Primers; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; C074119:heat-shock protein 65, Mycobacterium; D018833:Chaperonins