Detection of amplifiedChlamydia trachomatis DNA using a microtiter plate-based enzyme immunoassay

Abstract

A microtiter plate-based method to detect amplified DNA was developed. The method uses on biotin-labeled primer in the polymerase chain reaction (PCR) mixture. The labeled amplicon is bound to streptavidin-coated microtiter plates, denatured and hybridized to a digoxigenin-labeled probe. The specificity of the hybridization reaction was optimized by varying the temperature of the subsequent washing step and adding urea to the washing buffer. The digoxigenin label was detected using an enzyme immunoassay (EIA). This PCR-EIA was compared with a standard PCR assay that uses gel electrophoresis, blotting and hybridization to detect the amplicon, with isolation in cell culture, and with an antigen detection EIA (Chlamydiazyme) in the diagnosis of Chlamydia trachomatis infection in 309 female patients attending a sexually transmitted diseases outpatient clinic. The prevalence of Chlamydia trachomatis infection 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 assay 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% and 98.9% for the PCR-EIA, and 97.4% and 98.9% for the standard PCR assay. It is concluded that the PCR-EIA described is a fast, sensitive and specific method for detecting Chlamydia trachomatis in clinical specimens.

Chlamydia trachomatisGenotypes: Correlation with Clinical Manifestations of Infection and Patients' Characteristics

“…It is suggested that PCR assays especially detect more cases of low-grade, to cause more complaints of lower abdominal pain, and in men genotypes H and J produced more symptoms of dysuria and otherwise inapparent infections [34]. As a result, in our study some chlamydial infections remained undetected, especially urethral discharge.…”

“…For the nested PCR, 1 mL was used in a second PCR C. trachomatis, as described previously [30]. One specimen was obtained by using the specially designed collection kit of using the primers NLI and NRI [34]. Both primer sets were designed to amplify all known serovars of C. trachomatis.…”

“…Brunschwig Chemie, Amsterdam, The Netherlands]; 1 mM each primer; and 1.5 U AmpliTaq [Perkin-Elmer, Gouda, The NetherFor men two urethral specimens and for women two endocervical specimens were collected for testing for the presence of lands]). The first PCR was carried out with the primers NLO and NRO [34]. For the nested PCR, 1 mL was used in a second PCR C. trachomatis, as described previously [30].…”

Resurgence of Lymphogranuloma Venereum in Western Europe: An Outbreak ofChlamydia trachomatisSerovar L2Proctitis in The Netherlands among Men Who Have Sex with Men

“…Urine and rectal swabs, which were collected in 2SP medium, were used for PCR testing. Genotyping of the gene encoding the major outer membrane protein (MOMP) was performed by nested PCR and RFLP analysis [6]. Serum specimens were collected and analyzed for the presence of specific IgA and IgG antibodies to C. trachomatis by peptide EIA (SeroCT; Savyon Diagnostics).…”

“…Chronic inflammation of the lymphatic vessels, progressive lymph edema, and sclerosing fibrosis may cause severe disfiguring conditions, such as genital elephantiasis and esthiomene [3,4]. Genotyping C. trachomatis by nested PCR and restriction fragment-length polymorphism (RFLP) allows a reliable diagnosis [5][6][7].…”