Use of PCR to detect Entamoeba gingivalis in diseased gingival pockets and demonstrate its absence in healthy gingival sites

Abstract

Investigators using light microscopy have identified the protozoan parasite Entamoeba gingivalis from diseased gingival pockets for nearly 100 years. The objective of the present investigation was to develop a molecular biology approach for determining the presence of E. gingivalis in both diseased gingival pockets and healthy gingival sites. For this, a previously developed conventional polymerase chain reaction (PCR) was evaluated and a real-time polymerase chain reaction assay was developed. Paper points were inserted into the base of the sulcus of both diseased gingival pockets and healthy gingival sites. DNA was extracted using the QIAamp DNA mini kit, and subsequently analyzed using conventional and real-time PCR analysis. A previously described primer set specific for the small subunit ribosomal RNA gene (SSU rDNA) of E. gingivalis was used for the conventional PCR. For the real-time PCR, a primer set was designed to amplify a 135-bp fragment inside the SSU rDNA of E. gingivalis. A conventional PCR assay detected E. gingivalis in 27% of diseased gingival pockets. The real-time PCR using a different primer set detected protozoa in 69% of diseased pocket sites. Thus, the latter technique proved more sensitive for detection of E. gingivalis. No E. gingivalis were detected in any of the healthy gingival pocket sites using either type of PCR assay. Results support a concept that the presence of E. gingivalis is associated only with diseased gingival pocket sites. The newly described methodology may also serve to provide a novel eukaryotic cell marker of disease status in gingival pockets.