Use of polymerase chain reaction techniques and sodium dodecyl sulfate-polyacrylamide gel electrophoresis for differentiation of oral Lactobacillus species.
OBJECTIVE
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.


CONCLUSIONS
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.
Department of Stomatology, Faculty of Dentistry, Prince of Songkla University, Thailand. rawee.t@psu.ac.th
R
R
Teanpaisan
G
G
Dahlén
D000328:Adult; D002648:Child; D004269:DNA, Bacterial / Q000032:analysis; D015257:DNA-Cytosine Methylases; D015252:Deoxyribonucleases, Type II Site-Specific; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D007778:Lactobacillus / Q000145:classification* / Q000302:isolation & purification; D007779:Lactobacillus acidophilus / Q000302:isolation & purification; D007780:Lacticaseibacillus casei / Q000302:isolation & purification; D052197:Limosilactobacillus fermentum / Q000302:isolation & purification; D048191:Lactiplantibacillus plantarum / Q000302:isolation & purification; D052201:Lacticaseibacillus rhamnosus / Q000302:isolation & purification; D009055:Mouth / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases