Applications of loop-mediated isothermal amplificaton methods (LAMP) for identification and diagnosis of mycotic diseases: Paracoccidioidomycosis and Ochroconis gallopava infection

Abstract

Loop-mediated isothermal amplification (LAMP) methods are now useful for the detection of a specific gene in infectious diseases, genetic diseases, and/or genetic disorders in the large number of medical fields, and it was recently introduced to fungal investigation. It is characterized by the use of four different primers specifically designed to recognize six distinct regions of the target gene, and the reaction process proceeds at a constant temperature using strand displacement reaction. Quickness and simplicity is the advantage of the method. Amplification and detection of gene can be completed in a single step, by incubating the mixture of samples, primers, DNA polymerase with strand displacement activity and substrates at a constant temperature. The method was applied to two fungal infections; paracoccidioidomycosis (PCM), a deep mycosis caused by Paracoccidioides brasiliensis and Ochroconis gallopava infection. For PCM a combination of F3, B3, FIP, and BIP primers designed from the partial sequence of P. brasiliensis gp43 gene was used. The PCR products amplified by the primer set; F3 and B3 showed species specificity for P. brasiliensis and the detection limit of the PCR was 100 fg of fungal genomic DNA. The specific DNA banding pattern of P. brasiliensis was detected in the clinical and nine-banded armadillo derived isolates, paraffin-embedded tissue sample or sputum from PCM patient. LAMP method was used also for the identification of O. gallopava by using species-specific primer sets based on the D1/D2 domain of the LSU rDNA sequence. The method successfully detected the gene from both fungal DNA derived from brains and spleens of experimentally-infected mice with O. gallopava and environmental isolates. In conclusion, LAMP method for PCM and O. gallopava seemed to be useful for identification, diagnosis or retrospective study with advantage in the quickness and simplicity procedure, but require strictly-controlled environments. © 2010 Springer-Verlag Berlin Heidelberg.