Rapid and simple detection of eight major periodontal pathogens by the loop-mediated isothermal amplification method.
Loop-mediated isothermal amplification (LAMP) was applied to develop a rapid and simple detection system for eight periodontal pathogens: Aggregatibacter (Actinobacillus) actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola and Tannerella forsythia. Primers were designed from the 16S ribosomal RNA gene for each pathogen, and the LAMP amplified the targets specifically and efficiently under isothermal condition at 64 degrees C. To simplify the manipulation of LAMP examination, boiled cells and intact cells suspended in phosphate-buffered saline (PBS) were tested as templates besides extracted DNA template. The detection limits were 1-10 cells per tube using extracted DNA template. However, LAMP methods using boiled cells and intact cells required 10-100 and 100-1000 cells per tube, respectively. LAMPs for A. actinomycetemcomitans, P. gingivalis and P. intermedia were then applied to clinical plaque samples, and the method demonstrated equal or higher sensitivity compared with the conventional real-time PCR method. These findings suggest the usefulness of the LAMP method for the rapid and simple microbiological diagnosis of periodontitis, and the possibility of LAMP examination without the DNA extraction step.
Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama, Japan.
Junko
J
Miyagawa
Hiroshi
H
Maeda
Toshimitsu
T
Murauchi
Susumu
S
Kokeguchi
Kokoro
K
Yamabe
Ichiro
I
Tanimoto
Fusanori
F
Nishimura
Kazuhiro
K
Fukui
Shogo
S
Takashiba
D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D003773:Dental Plaque / Q000382:microbiology; D020459:Genes, rRNA / Q000235:genetics; D006089:Gram-Negative Anaerobic Bacteria / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010510:Periodontal Diseases / Q000382:microbiology*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S