Rapid detection of the cariogenic pathogens Streptococcus mutans and Streptococcus sobrinus using loop-mediated isothermal amplification.
Oral Microbiol Immunol
; 22(6): 361-8, 2007 Dec.
Article
in En
| MEDLINE
| ID: mdl-17949337
ABSTRACT
INTRODUCTION:
Streptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries in humans. In this study, we developed a rapid, sensitive method for detecting these major cariogenic pathogens using loop-mediated isothermal amplification (LAMP). The assay procedure is quite simple the amplification is carried out in a single tube under isothermal conditions at 63 degrees C, and the result can be obtained in less than 1 h.METHODS:
Initially, a set of six primers was designed by targeting S. mutans-specific and S. sobrinus-specific regions, identified using the genomic subtractive hybridization technique. We evaluated the specificities and sensitivities of these assays. Furthermore, we detected and quantified these bacteria in saliva and carious dentin from eight children.RESULTS:
The sensitivities of the S. mutans-specific and S. sobrinus-specific LAMP methods, examined using agarose gel electrophoresis, were each one cell for a 30-min reaction. The detection limits using real-time turbidimetry analysis were 1 to 10(7) cells (3.28 x 10(1) to 3.28 x 10(8) fg S. mutans template DNA) per reaction tube and 1 to 10(5) cells (2.72 x 10(3) to 2.72 x 10(8) fg S. sobrinus template DNA) per reaction tube. Using these assays, we detected and quantified these cariogenic bacteria for evaluation of the LAMP assay for clinical diagnosis.CONCLUSIONS:
Our results suggest that the LAMP-based assay in combination with subtractive hybridization is valuable for preparing species-specific primers for closely related species. Furthermore, the LAMP-based assay will be a useful tool for the rapid and sensitive prediction of dental caries.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Streptococcus mutans
/
Streptococcus sobrinus
/
Nucleic Acid Amplification Techniques
/
Dental Caries
Type of study:
Diagnostic_studies
Limits:
Child
/
Child, preschool
/
Female
/
Humans
/
Male
Language:
En
Journal:
Oral Microbiol Immunol
Journal subject:
ALERGIA E IMUNOLOGIA
/
MICROBIOLOGIA
/
ODONTOLOGIA
Year:
2007
Document type:
Article
Affiliation country:
Japan