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

Artikel i vetenskaplig tidskrift
Författare R Teanpaisan
Gunnar Dahlén
Publicerad i Oral microbiology and immunology
Volym 21
Nummer/häfte 2
Sidor 79-83
ISSN 0902-0055
Publiceringsår 2006
Publicerad vid Institutionen för odontologi
Sidor 79-83
Språk en
Länkar dx.doi.org/10.1111/j.1399-302X.2006...
Ämnesord Adult, Child, DNA, Bacterial, analysis, Deoxyribonucleases, Type II Site-Specific, diagnostic use, Electrophoresis, Polyacrylamide Gel, methods, Humans, Lactobacillus, classification, isolation & purification, Lactobacillus acidophilus, isolation & purification, Lactobacillus casei, isolation & purification, Lactobacillus fermentum, isolation & purification, Lactobacillus plantarum, isolation & purification, Lactobacillus rhamnosus, isolation & purification, Mouth, microbiology, Polymerase Chain Reaction, methods, Polymorphism, Restriction Fragment Length, RNA, Ribosomal, 16S, analysis, Sequence Analysis, DNA, Site-Specific DNA Methyltransferase (Cytosine-Specific), diagnostic use
Ämneskategorier Oral mikrobiologi

Sammanfattning

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.

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