Development of a Colorimetric Microtiter Plate Hybridization Assay To Detect Rt/Pcr Products of Mycobacterium Zeprae
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Date
1997-06
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Addis Ababa University
Abstract
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.
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Biology