title: Comparison of pulsed-field gel electrophoresis (PFGE) and two different polymerase chain reactions (PCRs) for species identification of Borrelia burgdorferi sensu lato strains.
abstract: The purpose of the present study was to compare the findings of three different molecular-biological methods for Borrelia burgdorferi sensu lato species identification: (i) large DNA fragments pattern obtained with Mlul restriction endonuclease and separated with pulsed-field gel electrophoresis (PFGE); (ii) polymerase chain reaction (PCR), the region inside the 16S rRNA gene multiplied with species-specific primers; and (iii) PCR, the interspace region between the 5S and 23S rRNA genes amplified, the PCR product restricted with Msel restriction endonuclease and fragments separated in polyacrylamide gel. Forty-eight Borrelia strains isolated from diverse clinical materials and two tick strains were analyzed. Each of the 50 isolates analyzed by PFGE was found to be a single species: 30 B. afzelii, 14 B. garinii, and 6 B. burgdorferi sensu stricto. PCR amplification of 16S rRNA with species-specific primers revealed a single species in 41/50 samples and in nine samples two species were detected. PCR of the 5S-23S interspace region restricted with MseI restriction endonuclease detected a single species in 48/50 samples and a mixture of two species was found in 2/50 samples. In all cases where a single species was identified using PCR the species was in accordance with the PFGE result, and in all cases where a mixture of two species was identified by PCR one of the species was the same as that detected by PFGE. Using a criterion of complete concordance of the results a significant difference in species identification was found when PFGE and the 16S rRNA PCR were compared (p = 0.0026), but not between 5S-23S interspace PCR and PFGE (p = 0.4949) or between 16S rRNA and 5S-23S interspace PCRs (p = 0.0552). PCR assays were faster and easier to perform than PFGE for Borrelia species identification, however PFGE remains a standard procedure for analyzing isolates and demonstrating heterogeneity within species.
authors.affiliation: Institute of Microbiology and Immunology, Medical Faculty, Ljubljana, Slovenia.
authors.forename: Janez
authors.initials: J
authors.lastname: Simenc
authors.forename: Eva
authors.initials: E
authors.lastname: Ruzić-Sabljić
authors.forename: Franc
authors.initials: F
authors.lastname: Strle
mesh_terms: D000818:Animals; D025065:Borrelia burgdorferi / Q000145:classification / Q000235:genetics; D015748:Borrelia burgdorferi Group / Q000145:classification* / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D018740:Genetic Heterogeneity; D006801:Humans; D008193:Lyme Disease / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017524:Slovenia; D013045:Species Specificity; D013987:Ticks / Q000382:microbiology*
chemical_list: D012336:RNA, Ribosomal, 16S