PCR-based detection of mycobacteria in paraffin embedded specimens: A molecular technique on its way into routine-diagnostics

Abstract

PCR is a unique technology for sensitive detection of mycobacterial DNA-sequences in cases where no fresh material for classical analyses can be obtained. We describe the set up of an optimized protocol and our experience of 154 cases analyzed during the last two years. In 35,7% of the cases the quality of the extracted DNA had been too low to obtain any information. In 33,8% of the cases with suitable DNA we did not amplify mycobacterial DNA. In 30,5% of the cases we detected mycobacterial DNA: 63,8% of those belonged to the M. tuberculosis-complex and 36,2% were non-tuberculous mycobacteria (NTM). Fragmentation of the DNA extracted from clinical samples on the one hand and the rigid cell wall of mycobacteria on the other hand are obstacles to detect the DNA of these micro-organisms by PCR in paraffin-embedded tissues. The introduction of heat / cold treatments during DNA-extraction improved the detection of mycobacterial DNA by breaking the mycobacterial cell walls although contributing to further DNA-fragmentation. Within its limitations, like the quality of DNA from paraffin embedded specimens and the large efforts necessary to control contaminations, PCR represents a powerful additional tool for routine diagnostics of mycobacterial infections.