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.
- DNA extraction
- polymerase chain reaction
- IFI, invasive fungal infection
- PCR, polymerase chain reaction
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This work was undertaken by Dr P J Paterson, Mrs S Seaton, Dr J McLaughlin, and Dr C C Kibbler with the Royal Free Hampstead NHS Trust who received a proportion of its funding from the NHS Executive; the views expressed in this publication are those of the authors and not necessarily those of the Trust or the NHS Executive.
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