Quantitative Detection of Mycobacterium Ieprae in Clinical Specimen by the Polymerase Chain Reactio
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Date
1995-09
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Addis Ababa University
Abstract
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.
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Biology