DETECTION OF AMPLIFIED CHLAMYDIA-TRACHOMATIS DNA USING A MICROTITER PLATE-BASED ENZYME-IMMUNOASSAY

A microtiter plate-based method to detect amplified DNA was developed. The method uses one biotin-labeled primer in the polymerase chain rea ction (PCR) mixture. The labeled amplicon is bound to streptavidin-coa ted microtiter plates, denatured and hybridized to a digoxigenin-label ed probe. The specificity of the hybridization reaction was optimized by varying the temperature of the subsequent washing step and adding u rea to the washing buffer. The digoxigenin label was detected using an enzyme immunoassay (EIA). This PCR-EIA was compared with a standard P CR assay that uses gel electrophoresis, blotting and hybridization to detect the amplicon, with isolation in cell culture, and with an antig en detection EIA (Chlamydiazyme) in the diagnosis of Chlamydia trachom atis infection in 309 female patients attending a sexually transmitted diseases outpatient clinic. The prevalence of Chlamydia trachomatis i nfection as determined by isolation in cell culture, EIA, PCR-EIA and standard PCR assay was 9.1 %, 8.7 %, 12.3 %, and 12.9 %, respectively. Compared with results of a reference set of confirmed-positive cases (defined by a positive result in two or more independent assays after analysis of discrepancies), the sensitivity and specificity was 71.1 % and 99.6 % for cell culture, 65.8 % and 99.3 % for the EIA, 92.1 % an d 98.9 % for the PCR-EIA, and 97.4 % and 98.9 % for the standard PCR a ssay. It is concluded that the PCR-EIA described is a fast, sensitive and specific method for detecting Chlamydia trachomatis in clinical sp ecimens.