TY - JOUR T1 - Development of molecular methods for the identification of aspergillus and emerging moulds in paraffin wax embedded tissue sections JF - Molecular Pathology JO - Mol Pathol SP - 368 LP - 370 DO - 10.1136/mp.56.6.368 VL - 56 IS - 6 AU - P J Paterson AU - S Seaton AU - J McLaughlin AU - C C Kibbler Y1 - 2003/12/01 UR - http://mp.bmj.com/content/56/6/368.abstract N2 - Background/Aims: Invasive infection with emerging moulds is increasing in incidence and reliable methods for speciating these organisms in tissue sections need to be developed. Methods: Two methods for extracting fungal DNA from paraffin wax embedded tissue sections, based on the TaKaRa DEXPAT™ kit and QIAamp® DNA mini kit, were optimised and compared. DNA was amplified by PCR using pan-fungal probes, and detected by Southern blot hybridisation using a high stringency method with a probe specific for Aspergillus fumigatus and A flavus. Results: The method based on the TaKaRa DEXPAT kit, with additional steps using lyticase and ethanol precipitation, was superior. Less than 10 conidia were detectable using spiked samples and a positive result was obtained with 100% of clinical samples known to be culture positive for A fumigatus. Other moulds could be identified by using species specific probes or by sequencing PCR products. Conclusions: The method based on the TaKaRa DEXPAT kit could detect less than 10 conidia/sample. The method allowed accurate identification of A fumigatus and A flavus and other species could be identified using species specific probes or by DNA sequencing. These methods will provide a valuable diagnostic tool for both patient management and future antifungal and epidemiological studies. ER -