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Development of a loop-mediated isothermal amplification assay for detecting Porphyromonas endodontalis
Eugene Joseph Whitaker1, Leena N Shah2, Ivan R Miloradovic2
1Department of Restorative Dentistry, Temple University School of Dentistry, Philadelphia, PA, USA.
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.
Insights
A new loop-mediated isothermal amplification (LAMP) assay was developed to detect the endodontic pathogen Porphyromonas endodontalis. This highly sensitive and specific assay shows promise for chairside diagnostics.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Loop-mediated isothermal amplification (LAMP) is a powerful technique for detecting microorganisms.
- Existing LAMP assays cover some endodontic pathogens like Enterococcus faecalis and Fusobacterium nucleatum.
- Porphyromonas endodontalis, a significant endodontic pathogen, currently lacks a specific LAMP assay.
Purpose of the Study:
- To develop and evaluate a novel LAMP assay for the specific detection of Porphyromonas endodontalis.
- To establish the sensitivity and specificity of the developed LAMP assay.
Main Methods:
- Identified a unique gene segment (DPP11) of P. endodontalis using BLAST.
- Designed six primers for the LAMP assay.
- Evaluated the assay's performance using purified P. endodontalis DNA and extracted DNA from various oral bacteria.
Main Results:
- The developed LAMP assay demonstrated 82% sensitivity and 90% specificity for P. endodontalis.
- The assay was tested on pure cultures and diluted DNA samples from P. endodontalis and other oral bacteria.
- Results were analyzed using contingency tables.
Conclusions:
- The developed LAMP assay is a highly sensitive and specific molecular tool.
- This assay has the potential to serve as a rapid chairside diagnostic method for P. endodontalis infections.
