Quantitation of Bacteroides forsythus in subgingival plaque comparison of immunoassay and quantitative polymerase chain reaction.
Our objective was to compare three methods (enzyme-linked immunosorbent assay [ELISA], endpoint and quantitative polymerase chain reaction [E-PCR and Q-PCR]) for detection and quantitation of Bacteroides forsythus in 56 plaque samples from seven subjects with progressive periodontal disease. Samples collected in buffer were pelleted and resuspended in 500 microl of water. Fifty microl aliquots were removed for an ELISA performed on bacteria or plaque immobilized on 96-well plates and probed with B. forsythus specific antibody. An occurrence of 3.7+/-0.6 x 10(4) or more bacteria were detected by ELISA in pure culture; 26 of 54 plaque samples were positive, two samples could not be analyzed. Samples for PCR were autoclaved for 10 min prior to use. The detection level of E-PCR using primers specific for B. forsythus 16S rRNA was 200 cells and 42 out of 56 samples were positive based on ethidium bromide stained agarose gels. Q-PCR using the same primers combined with a nested fluorescent oligonucleotide probe detected 10+/-0.32 bacteria in pure culture; 43 of 56 plaque samples were positive. The ELISA and Q-PCR obtained identical results with 36 of the 54 samples assayed; there were one false positive and 17 false negative ELISA results using Q-PCR as standard. The positive proportions of plaque samples were almost the same for E-PCR and Q-PCR. We conclude that the PCR methods are more appropriate for a multicenter study because of greater sensitivity and convenience of sample transportation from clinics to a central laboratory.
Shelburne
C E
CE
3M Company, St. Paul, MI 55144, USA. ceshelburne@mmm.com
Prabhu
A
A
Gleason
R M
RM
Mullally
B H
BH
Coulter
W A
WA
D001439:Bacteroides / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D001442:Bacteroides Infections / Q000382:microbiology; D003773:Dental Plaque / Q000382:microbiology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D010510:Periodontal Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*