Rapid detection of the cariogenic pathogens Streptococcus mutans and Streptococcus sobrinus using loop-mediated isothermal amplification.
BACKGROUND
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.
Nagashima
S
S
Division of Community Oral Health Science, Kyushu Dental College, Kitakyushu, Japan.
Yoshida
A
A
Ansai
T
T
Watari
H
H
Notomi
T
T
Maki
K
K
Takehara
T
T
D002648:Child; D002675:Child, Preschool; D017931:DNA Primers; D003731:Dental Caries / Q000382:microbiology*; D003804:Dentin / Q000382:microbiology; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012463:Saliva / Q000382:microbiology; D013045:Species Specificity; D013295:Streptococcus mutans / Q000235:genetics / Q000302:isolation & purification*; D016986:Streptococcus sobrinus / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers