Rapid detection of Actinobacillus actinomycetemcomitans using a loop-mediated isothermal amplification method.
BACKGROUND
Actinobacillus actinomycetemcomitans has been implicated in the etiology of aggressive periodontitis. In this study, we applied a novel nucleic acid amplification method, called loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions, allowing the rapid detection of A. actinomycetemcomitans.


METHODS
We designed the primers for detecting A. actinomycetemcomitans and evaluated the specificity and sensitivity of the assay.


RESULTS
The LAMP primers used in this study successfully amplified serotypes a-e of A. actinomycetemcomitans, while other oral bacteria were not amplified. By measuring the precipitation of magnesium pyrophosphate, we could quantify the chromosomal DNA of A. actinomycetemcomitans. The detection limits using the real-time turbidimetry analysis were 5.8 x 10(2)-5.8 x 10(7) copies of A. actinomycetemcomitans template DNA per reaction tube. In addition, the LAMP assay was used for the rapid detection of A. actinomycetemcomitans in clinical specimens from eight individuals. The results with the LAMP method were similar to those using conventional polymerase chain reaction.


CONCLUSIONS
Our results suggest that the LAMP-based assay is very useful for the rapid detection of A. actinomycetemcomitans.
Osawa
R
R
Division of Community Oral Health Science, Kyushu Dental College, Kokurakita-ku, Kitakyushu, Japan.
Yoshida
A
A
Masakiyo
Y
Y
Nagashima
S
S
Ansai
T
T
Watari
H
H
Notomi
T
T
Takehara
T
T
D000328:Adult; D000368:Aged; D016976:Aggregatibacter actinomycetemcomitans / Q000302:isolation & purification*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis*; D003773:Dental Plaque / Q000382:microbiology; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010514:Periodontal Pocket / Q000382:microbiology; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013696:Temperature
D004269:DNA, Bacterial