Polymerase chain reaction amplifying DNA coding for species-specific rRNA of Mycobacterium leprae.
The sensitivity of the polymerase chain reaction (PCR) on the DNA coding for the species-specific fragment of 16S rRNA of Mycobacterium leprae studied on mouse foot pad harvests and human skin biopsies varies widely between 1 and 3 x 10(4) organisms. This is probably the result of variations in the proportions of organisms with sufficiently intact DNA suitable for PCR. Preserving human skin biopsies for 3 weeks at an ambient temperature even after boiling for 6 minutes gives rise to a 10-fold decrease in sensitivity. Fixation of tissues in formol 10% or Lowy fixative or preserving in Dubos OAA broth is very harmful to the PCR, mainly due to the enhancement of an inhibitory effect on the PCR reaction. For preservation, the best choice at the moment seems to be alcohol 70%. Sample preparation of five cycles of freeze-boiling is simple and generally more efficient than proteinase K treatment and DNA extraction.
Pattyn
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Institute for Tropical Medicine, Antwerp, Belgium.
Ursi
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Ieven
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Raes
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Jamet
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D000818:Animals; D001483:Base Sequence; D001706:Biopsy; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D019286:Endopeptidase K; D005615:Freezing; D006358:Hot Temperature; D006801:Humans; D007918:Leprosy / Q000382:microbiology*; D051379:Mice; D008969:Molecular Sequence Data; D009166:Mycobacterium leprae / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011309:Preservation, Biological; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics*; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000378:metabolism; D012867:Skin / Q000382:microbiology; D013045:Species Specificity
D003470:Culture Media; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012697:Serine Endopeptidases; D019286:Endopeptidase K