Development of a loop-mediated isothermal amplification assay for detecting Porphyromonas endodontalis.

Abstract

Context: Loop-mediated isothermal amplification (LAMP) may be used in the future to detect infecting microorganisms. LAMP assays exist for the endodontic pathogens Enterococcus faecalis and Fusobacterium nucleatum, but not yet for Porphyromonas endodontalis.

Aim: To develop a LAMP assay for detecting P. endodontalis.

Settings and design: It was an in vitro benchtop study.

Subjects and methods: The National Center for Biotechnology Information GenBank Basic Local Alignment Search Tool was used to identify a segment of the dipeptidyl peptidase 11 (DPP11) gene unique to P. endodontalis. A primer design tool was used to generate six primers required for developing the LAMP assay. WarmStart Colorimetric LAMP 2X Master Mix was used to evaluate the LAMP assay, using purified P. endodontalis DNA as a control.

Statistical analysis used: Statistical parameters for sensitivity and specificity.

Results: The assay was performed in triplicate on pure DNA from P. endodontalis and P. gingivalis and on the DNA that was extracted from P. endodontalis, P. gingivalis, F. nucleatum, and E. faecalis cells and diluted two-fold from 1/2 to 1/256. Assays for the diluted samples were performed in triplicate, and the contingency tables indicated the LAMP assay to be 82% sensitive and 90% specific for P. endodontalis.

Conclusions: LAMP assay could be a highly sensitive and specific chairside detection method for P. endodontalis.