HOPE-Fixation Enables Improved PCR-Based Detection and Differentiation of Mycobacterium tuberculosis Complex in Paraffin-Embedded Tissues

• Published in: Pathology, research and practice research and practice, 2003, Vol.199 (9), p.619-623
• Main Authors: Sen Gupta, R., Hillemann, D., Kubica, T., Zissel, G., Müller-Quernheim, J., Galle, J., Vollmer, E., Goldmann, T.
• Format: Article
• Language: English
• Subjects: Animals; DNA, Bacterial - analysis; Female; Fixation; Fixatives; Granuloma - immunology; Granuloma - microbiology; Granuloma - pathology; HOPE ( Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect); Mice; Mice, SCID; Mycobacteria diagnosis; Mycobacterium bovis - isolation & purification; Mycobacterium bovis - pathogenicity; Mycobacterium bovis - physiology; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - isolation & purification; Paraffin Embedding; Polymerase Chain Reaction; Real-time-PCR; Sequence Analysis, DNA; Spoligotyping; Tissue Fixation - methods; Tuberculosis, Pulmonary - diagnosis; Tuberculosis, Pulmonary - immunology; Tuberculosis, Pulmonary - microbiology
• ISSN: 0344-0338; 1618-0631
• DOI: 10.1078/0344-0338-00470

Standard PCR-based detection of mycobacterial DNA in paraffin-embedded specimens may lack sufficient sensitivity because of the degradation of nucleic acids caused by routinely used formalin fixation. Therefore, we set up an approach that aimed at improving the results by applying the novel HOPE-fixative in PCR-detection of mycobacteria in paraffin-embedded tissues. Comparison of PCR-results using DNA extracted from either HOPE- or formalin-fixed specimens in BCG-infected SCID-mice revealed a more than 100fold enhanced sensitivity for the HOPE-fixed material. Owing to the preservation of DNA from degradation in HOPE-fixed tissues, even differentiation within the M. tuberculosis complex was possible by spoligotyping. We therefore conclude that the HOPE-fixative is a useful tool for molecular pathology that enhances the sensitivity of PCR-based methods for the detection of pathogens in paraffin-embedded tissues compared to formalin-fixation. Owing to the better preserved DNA, improved differentiation of mycobacteria from archived materials is possible. These results promise new and a substantially wider range of possibilities in the field of molecular pathology.