In order to improve detection of Mycobacterium /eprae in clinical specimens, an ELISAbasedrndetection method for a 176 bp fragment generated by RT/PCR for the 16S rRl"lArnwas developed. Total extracted RNA was reverse transcribed to make cDNA, and wasrnsubjected to PCR amplification using one biotinylated primer. The products of PCRrnamplification were added to streptavidin-coated microtiter plates, denatured with NaOH,rnhybridized with a FITC labelled 16S rRNA oligonucleotide probe, then anti-FITCrnantibody conjugated with alkaline phosphatase was added, and finally subjected torncolorimetric detection using pNPP as a substrate to the alkaline phosphatase. Three typesrnof microtiter plate assays were investigated. Of these, one (indirect assay withoutrnblocking reagent) was confirmed to be simple, as well as sensitive for detecting M.rn/eprae in leprosy patients. This assay was highly specific as, no amplification wasrnobserved with skin biopsies from normal individuals or patients with skin diseases otherrnthan leprosy. After optimization of the assay, the sensitivity of the system was furtherrnassessed by using serially-diluted bacilli. It was very sensitive and detected as few as 10rnbacilli. By examining 61 tissue biopsies from leprosy patients, the assays sensitivity forrnclinical specimens was assessed. The assay detected M. /ep;'ae RT IPCR products inrn100% multibacillary patients, and in 80% of paucibacillary patients. In addition, sincern16S rRNA is rapidly degraded in dead cells, application of ELISA-based RT/PCR wouldrnbe most appropriate for the diagnosis of difficult cases harboring live bacteria. Suchrninformation would be important for further patient management.