R Teanpaisan1, G Dahlén. 1. Department of Stomatology, Faculty of Dentistry, Prince of Songkla University, Thailand. rawee.t@psu.ac.th
Abstract
BACKGROUND/AIMS: The genus Lactobacillus has been associated with dental caries in humans, although it is seldom speciated due to lack of simple and nonlaborious identification methods. A considerable heterogeneity among Lactobacillus species has been demonstrated. The purpose of this study was to develop simple methods combining restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S rRNA (16S rRNA gene PCR-RFLP) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the identification of 13 reference strains of Lactobacillus. METHODS: The 16S rRNA gene sequences were amplified by PCR using universal primers and digestion of PCR products with the restriction endonucleases, HpaII and HaeIII. The 16S rRNA gene PCR-RFLP is reproducible and has been proved to be useful for differentiating Lactobacillus strains to species level. Seventy-seven Lactobacillus isolates from a Thai population were used to show the applicability of the identification test. RESULTS: PCR-RFLP alone had limitations, because the RFLP patterns of Lactobacillus casei and Lactobacillus rhamnosus and of Lactobacillus acidophilus and Lactobacillus crispatus showed similar patterns; however, these could be differentiated by SDS-PAGE. Of the 77 isolates, 38 were identified as Lactobacillus fermentum, 25 as L. rhamnosus, 5 as Lactobacillus salivarius, 5 as L. casei, 3 as L. acidophilus and 1 as Lactobacillus plantarum. CONCLUSION: 16S rRNA gene PCR-RFLP, using HpaII and HaeIII, together with SDS-PAGE protein profiles could be an alternative method for the identification of oral Lactobacillus strains to species level, and may be applicable for large-scale studies on the association of Lactobacillus to dental caries.
BACKGROUND/AIMS: The genus Lactobacillus has been associated with dental caries in humans, although it is seldom speciated due to lack of simple and nonlaborious identification methods. A considerable heterogeneity among Lactobacillus species has been demonstrated. The purpose of this study was to develop simple methods combining restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S rRNA (16S rRNA gene PCR-RFLP) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the identification of 13 reference strains of Lactobacillus. METHODS: The 16S rRNA gene sequences were amplified by PCR using universal primers and digestion of PCR products with the restriction endonucleases, HpaII and HaeIII. The 16S rRNA gene PCR-RFLP is reproducible and has been proved to be useful for differentiating Lactobacillus strains to species level. Seventy-seven Lactobacillus isolates from a Thai population were used to show the applicability of the identification test. RESULTS: PCR-RFLP alone had limitations, because the RFLP patterns of Lactobacillus casei and Lactobacillus rhamnosus and of Lactobacillus acidophilus and Lactobacillus crispatus showed similar patterns; however, these could be differentiated by SDS-PAGE. Of the 77 isolates, 38 were identified as Lactobacillus fermentum, 25 as L. rhamnosus, 5 as Lactobacillus salivarius, 5 as L. casei, 3 as L. acidophilus and 1 as Lactobacillus plantarum. CONCLUSION: 16S rRNA gene PCR-RFLP, using HpaII and HaeIII, together with SDS-PAGE protein profiles could be an alternative method for the identification of oral Lactobacillus strains to species level, and may be applicable for large-scale studies on the association of Lactobacillus to dental caries.