Loop-mediated isothermal amplification method for the rapid detection of Enterococcus faecalis in infected root canals.
BACKGROUND
Enterococcus faecalis is a major pathogen in the etiology of apical periodontitis after root canal treatment. A loop-mediated isothermal amplification method, which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand-displacement activity, was developed for the rapid detection of E. faecalis in clinical specimens from root canals.


METHODS
Primers for detecting E. faecalis from the azoA gene were designed. The specificity of this assay was evaluated using various oral bacteria and the sensitivity was evaluated using serially diluted E. faecalis chromosomal DNA. In addition, loop-mediated isothermal amplification assays were applied to the rapid detection of E. faecalis from endodontic samples.


RESULTS
The loop-mediated isothermal amplification products amplified with the primer set were specific for E. faecalis. To confirm the specificity of the amplicon, the amplified products were digested with the restriction endonuclease Sau3AI. The lower detection limit of the E. faecalis primer set without the loop primer set was 10 microg/tube for a 50-min loop-mediated isothermal amplification reaction. Using loop primers increased the detection sensitivity by several orders of magnitude. Furthermore, E. faecalis was detected with the loop-mediated isothermal amplification assay in four root canals from 18 individuals and the detection results were consistent with those of conventional polymerase chain reactions.


CONCLUSIONS
These results indicate that the loop-mediated isothermal amplification assay is very useful for rapid detection of E. faecalis and diagnosis of endodontic infection.
Kato
H
H
Division of Community Oral Health Science, Kyushu Dental College, Fukuoka, Kyushu, Japan.
Yoshida
A
A
Ansai
T
T
Watari
H
H
Notomi
T
T
Takehara
T
T
D000293:Adolescent; D000328:Adult; D000368:Aged; D001483:Base Sequence; D002648:Child; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D003786:Dental Pulp Cavity / Q000382:microbiology*; D013293:Enterococcus faecalis / Q000235:genetics* / Q000302:isolation & purification*; D005260:Female; D005798:Genes, Bacterial; D016908:Gram-Positive Bacterial Infections / Q000382:microbiology*; D006358:Hot Temperature; D006801:Humans; D008297:Male; D008875:Middle Aged; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010485:Periapical Periodontitis / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D004259:DNA-Directed DNA Polymerase