Quantitative Detection of Mycobacterium Ieprae in Clinical Specimen by the Polymerase Chain Reactio

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Addis Ababa University


Detection of M.leprae by peR provides a tool for identification of organisms in clinical specimens. Genomic DNA and RNA were isolated using commercially obtained extracting solutions (DNA STAT 60Thl and RNA STAT 60ThI ). RNA was reverse transcribed to make cDNA and both genomic DNA and cDNA were subjected to PCR amplification. Products were electrophoresed through agarose gels, stained with ethidium bromide, blotted onto nylon membranes',hybridized with a 16S rRNA oligonucleotide probe and subjected to non-radioactive detection. Several sets of 16S rRNA primers were investigated. Of these, one set of primers (P2 and P3) en = bp # 68-90 and P3 = bp # 218-239) was confirmed to be specific for M.leprae by testing its ability to amplify a panel of 28 potentially cross-reacting species of mycobacteria, 8 related non-mycobacterial isolates and 2 nasopharyngeal commensal organisms. In addition no amplification was observed when skin biopsies from normal individuals or patients with skin diseases other than leprosy were tested. Another set of primers (PI and P3) (PI = bp # 9-28 and P3= bp # 218-239) was genus-specific. After optimization of the peR condition. the sensitivity of the system was assessed using M.lepraespecific primers and the detection limit was 23 bacteria. This technique, therefore. appears to meet the criteria of sensitivity and specificity and may serve as a useful tool for the diagnosis of leprosy. By examining a limited number of tissues from leprosy patients, we were able to detect peR signals in all MB (n= 5) and in 89 % (n= 9) of PB patients. We also quantitated numbers of organisms in these specimens by comparing the density of the peR signal of samples on hybridized Southern blots to that of serial dilutions of known number of organisms.