AN IMPROVED METHOD FOR THE SPECIES-SPECIFIC ASSESSMENT OF MYCOBACTERIA IN ROUTINELY FORMALIN-FIXED AND PARAFFIN-EMBEDDED TISSUES

A polymerase chain reaction (PCR) assay for the rapid and species-spec ific diagnosis of mycobacterial infections in paraffin-embedded clinic al specimens was developed using oligonucleotide primers to amplify a fragment of the DNA coding for the ribosomal 16S RNA of mycobacteria. The oligonucleotide primers amplified DNA from all 14 species of mycob acteria tested. By means of a reamplification protocol, as few as one to two mycobacteria could be detected in the presence of human DNA. Th e method of DNA isolation and amplification was applied on sections of routinely formalin-fixed and paraffin-embedded tissues. PCR for the b eta-actin gene served as a control for successful DNA isolation. Mycob acterial DNA could be detected in cases of mycobacterial infections. T he mycobacterial species was determined by additional sequencing of th e PCR fragment. This PCR method may be a powerful tool for the diagnos is of mycobacterial infections from histopathological material and for the assessment of those mycobacteria that cannot readily be cultured, such as Mycobacterium leprae.