Development of a Colorimetric Microtiter Plate Hybridization Assay To Detect Rt/Pcr Products of Mycobacterium Zeprae

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Addis Ababa University


In order to improve detection of Mycobacterium /eprae in clinical specimens, an ELISAbased detection method for a 176 bp fragment generated by RT/PCR for the 16S rRl"lA was developed. Total extracted RNA was reverse transcribed to make cDNA, and was subjected to PCR amplification using one biotinylated primer. The products of PCR amplification were added to streptavidin-coated microtiter plates, denatured with NaOH, hybridized with a FITC labelled 16S rRNA oligonucleotide probe, then anti-FITC antibody conjugated with alkaline phosphatase was added, and finally subjected to colorimetric detection using pNPP as a substrate to the alkaline phosphatase. Three types of microtiter plate assays were investigated. Of these, one (indirect assay without blocking reagent) was confirmed to be simple, as well as sensitive for detecting M. /eprae in leprosy patients. This assay was highly specific as, no amplification was observed with skin biopsies from normal individuals or patients with skin diseases other than leprosy. After optimization of the assay, the sensitivity of the system was further assessed by using serially-diluted bacilli. It was very sensitive and detected as few as 10 bacilli. By examining 61 tissue biopsies from leprosy patients, the assays sensitivity for clinical specimens was assessed. The assay detected M. /ep;'ae RT IPCR products in 100% multibacillary patients, and in 80% of paucibacillary patients. In addition, since 16S rRNA is rapidly degraded in dead cells, application of ELISA-based RT/PCR would be most appropriate for the diagnosis of difficult cases harboring live bacteria. Such information would be important for further patient management.