Detection of Mycobacterium avium subsp. paratuberculosis in formalin- fixed, paraffin embedded tissues of goats by IS900 PCR

摘要

The objective of the present study was to optimise a DNA extraction procedure for formalin-fixed, paraffin embedded tissue sections for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) by IS900 specific polymerase chain reaction (PCR) in cases of naturally occurring paratuberculosis in goats. A total of 60 paraffin blocks (33 from small intestine and 27 from mesenteric lymph nodes) with various grades of histological lesions were used in the study. Three DNA extraction procedures were evaluated and the results analysed by PCR. The PCR carried out directly on the supernatant of tissue pellets disrupted in PBS by beating with zirconium/silica beads or on following DNA purified from the supernatant using a DNeasy kit (Qiagen) did not yield consistent results. The combination of bead beating rehydrated tissue pellets in the lysis buffer provided in the DNeasy kit, overnight proteinase K digestion and subsequent DNA extraction and ethanol precipitation gave consistent results and were adopted for all paraffin sections thereafter. The overall sensitivity of the PCR was 76% of all the samples that included various grades of lesions showing none or few to abundant acid-fast bacilli (AFB). The sensitivity of the assay was over 86% (38/44) in all paraffin sections having clearly and easily demonstrable bacilli. 50% of the tissue sections with rarely detectable AFB were also positive in the PCR implying a high quality of the DNA obtained by the procedure. There was no significant difference between the sensitivity of the PCR analyses carried out on intestinal vs. mesenteric lymph node tissues. The results of this study suggest that IS900 PCR on formalin-fixed, paraffin embedded histological sections is a practical and important tool for confirming diagnosis of paratuberculosis in goats, where fresh tissues for bacterial culture or PCR are not available due to cold storage and shipment problems. The procedure also can be adopted on the archived tissues.