A colorimetric detection system for Calymmatobacterium granulomatis

Objective: To incorporate the first polymerase chain reaction (PCR) assay f ur Calymmatobacterium granulomatis into a colorimetric detection system for use in routine diagnostic laboratories. Methods: A capture oligonucleotide specific for the Klebsiella phoE gene wa s covalently linked to tosyl activated magnetic beads. Biotinylated phoE PC R products obtained from 14 positive specimens from patients with donovanos is and isolates of Klebsiella pneumoniae, K rhinoscleromatis, and K ozaenae were cleaved with HaeIII for the purpose of differentiation, captured by t he prepared beads, and subjected to standard EIA detection methodology. Eig ht samples from unrelated genital conditions underwent the same procedure. It was anticipated from the sequence data that the biotinylated fragment wo uld be cleaved from the capture oligonucleotide target region in the three Klebsiella phoE products (that is, a negative colorimetric result) while th e entire fragment of interest would remain intact in the positive C granulo matis phoE products (that is, a positive colorimetric result). Results: All 14 positive specimens from patients with donovanosis gave stro ng colorimetric readings with this detection system. Isolates of K pneumoni ae, K rhinoscleromatis, K ozaenae and the eight specimens from unrelated ge nital conditions were negative. Conclusion: The successful development of a colorimetric detection system f or C granulomatis incorporating two levels of specificity enables the molec ular diagnosis of this condition to be undertaken by routine diagnostic lab oratories. This should have an important rule in the Australian government' s campaign to eradicate donovanosis by 2003 though the rest still needs to undergo trials and be validated using a larger number of samples from geogr aphically diverse parts of the world in order to ascertain the generalisabi lity of the methodology.