Last update:
02-Nov-2000

Arch Hellen Med, 17(2), March-April 2000, 171-179

ORIGINAL PAPER

Differential diagnosis of filamentous fungi by unitary digestion
of polymerase chain reaction products

M.E. KAMBOURIS, A. VELEGRAKI, N.J. LEGAKIS
Department of Microbiology, Mycology Reference Laboratory,
Medical School, University of Athens, Greece

OBJECTIVE The timely and accurate diagnosis of filamentous fungi is of paramount importance in improving the prognosis of the caused fungal infections.
METHOD Using existing protocols for fungal DNA extraction from clinical samples, a molecular diagnostic method was developed based on the ITS-1/4 specific polymerase chain reaction (PCR) for fungal detection, followed by restriction enzyme analysis of the PCR product by the restriction endonucleases Msp I, Hinf I, Hae III and Eco RI.
RESULTS The electrophoretic patterns readily distinguished the examined strains (belonging to the genera Acremonium, Penicillium, Rhizopus, Mucor, Trichoderma, Paecilomyces and the species A. fumigatus, A. carneus, A. terreus, P. viridicatum, F. oxysporum, F. solanii) down to genus level, and in exceptional cases to the species level. The specific sensitivity of the method was established at 20 A. fumigatus conidia per mL of whole blood, by spiking 250 μL of whole blood with enumerated conidial suspension, and ultimately by digesting with Eco RI the ITS-1/4 product, which resulted from the extraction and amplification procedures.

Key words: Diagnostic PCR, Filamentous fungi, Restriction enzyme analysis.