A rapid chemiluminescent DNA hybridization assay for the detection of Chlamydia trachomatis

Abstract

With an estimated 3-4 million new cases per year, human infections from Chlamydia trachomatis are probably the most prevalent sexually transmitted disease (STD) in the United States. Diagnosis of Chlamydia is usually conducted by tissue culture methods. Direct immunofluorescence and ELISA tests have become available, but there remains a need for a test with better specificity and sensitivity. In response to this need, we have developed a rapid DNA hybridization assay using synthetic oligonucleotide probes to detect the presence of the Chlamydia trachomatis specific 7.4 kb plasmid. The assay involves solution phase hybridization of unlabelled probes, rapid capture of the probe-target duplex onto a microtitre dish surface, a new signal amplification technique that employs chemically cross-linked oligonucleotides, and an alkaline phosphatase labelled probe. Signal is obtained by reacting the labelled probe-target complex with an enzyme triggerable dioxetane substrate. Detection of the chemiluminescent output is performed either with a luminometer or by exposure to instant film. All 15 serovars of Chlamydia trachomatis react positively, while organisms known to co-inhabit the human urogenital tract react negatively.

Secretion of proinflammatory cytokines by epithelial cells in response to Chlamydia infection suggests a central role for epithelial cells in chlamydial pathogenesis.

“…C. trachomatis serovar L2/434/Bu was propagated in L929 suspension cultures, C. trachomatis serovars D/UW3/ Cx and I/UW12/Ur, and C. psittaci AP1 were propagated in L929 cell monolayers, as described previously (31). Briefly, cultures were grown in RPMI growth medium (RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 60 g/ml vancomycin, and 10 g/ml gentamicin) at 37 Њ C in 95% air/5% CO 2 .…”

“…Infected monolayers were detached by scraping, and then pelleted and sonicated to lyse the host cells. Cell debris was removed by differential centrifugation as described previously (31). Chlamydial EBs were pelleted, resuspended in isotonic sucrose-phosphate-glutamate buffer (31), and frozen at Ϫ 80 Њ C. Infectious titers were determined by titration on L929 cell monolayers and staining with a FITC-labeled monoclonal antibody against chlamydial LPS (Meridian Diagnostics, Inc., Cincinnati, OH), and are expressed in inclusion-forming units (IFUs).…”

Clinical applications of luminescent assays for enzymes and enzyme labels

“…The process of enzyme-catalyzed chemiluminescent decomposition of 1 ,Zdioxetanes with suitable appendant groups in clinical assays has been recently described (19,20). 3-(2'-Spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD) is a substrate which can be activated to chemiluminescence in the presence of the enzyme alkaline phosphatase in aqueous buffers without addition of other co-reagents.…”

Advances in nucleic acid-based detection methods

“…Another chemiluminescent compound is phenylphosphatesubstituted dioxetane (84). This compound undergoes conversion to a luminescent form in the presence of alkaline phosphatase; it is reportedly 10 times more sensitive than colorimetric detection with o-phenylenediamine or horseradish peroxidase (19). Chlorine-and bromine-substituted dioxetanes have also been described (12); these improve chemiluminescent detection by eliminating nonenzymatically activated chemiluminescence, and they have short enzyme activation times.…”