Comparison of pulsed-field gel electrophoresis (PFGE) and two different polymerase chain reactions (PCRs) for species identification of Borrelia burgdorferi sensu lato strains.
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.
Simenc
Janez
J
Institute of Microbiology and Immunology, Medical Faculty, Ljubljana, Slovenia.
Ruzić-Sabljić
Eva
E
Strle
Franc
F
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*
D012336:RNA, Ribosomal, 16S