Detection and identification of mycobacteria in formalin-fixed, paraffin-embedded tissues by nested PCR and restriction enzyme analysis.
A novel assay based on a nested PCR and restriction enzyme analysis of the PCR products was developed for the rapid detection and identification of Mycobacterium bovis and M. avium-M. intracellulare species in formalin-fixed, paraffin-embedded tissue (PET) specimens. On the basis of the nucleotide sequence data obtained in the present study, general nested primers were constructed to amplify a 424-bp segment of the gene encoding the 65-kDa surface antigen of mycobacteria. The nested PCR assay proved to be highly sensitive, since as little as 5 to 10 fg of extracted mycobacterial DNA was detected. The safety of the assay as a routine method for the diagnosis of M. bovis and M. avium-M. intracellulare in PET specimens was provided by taking various precautions. In order to prevent false positivity, specific tools and procedures were applied. To detect false-negative results and assess the efficiency of the PCR, an internal standard molecule of amplification was constructed. The digestion of the amplicons with the restriction endonuclease Sau96-I allowed the identification of M. bovis and M. avium-M. intracellulare in a large number of clinical specimens. The present results indicate that PCR combined with an internal control of amplification and restriction enzyme analysis of the amplicons provides a rapid, sensitive, and reliable method for routine diagnostic laboratories to detect and identify M. bovis and M. avium-M. intracellulare in PET specimens.
Bascuñana
C R
CR
National Veterinary Institute, Uppsala, Sweden.
Belák
K
K
D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis*; D005557:Formaldehyde; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics / Q000302:isolation & purification*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D016707:Tissue Fixation
D017931:DNA Primers; D004269:DNA, Bacterial; D005557:Formaldehyde