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 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 specifi...

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Bibliographic Details
Published in:European journal of clinical microbiology & infectious diseases 1994-09, Vol.13 (9), p.732-740
Main Authors: Ossewaarde, J M, Rieffe, M, van Doornum, G J, Henquet, C J, van Loon, A M
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Chlamydia Infections - diagnosis
Chlamydia trachomatis
Chlamydia trachomatis - genetics
Chlamydia trachomatis - isolation & purification
DNA, Bacterial - isolation & purification
Female
Humans
Immunoenzyme Techniques
Molecular Sequence Data
Oligonucleotides
Polymerase Chain Reaction
Sensitivity and Specificity
Vaginal Smears
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Summary: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.
ISSN:0934-9723
DOI:10.1007/bf02276056