Preliminary clinical evaluation of SCAR-type molecular markers for the identification of the section Fumigati, A. flavus, and A. niger in bronchoalveolar lavage samples

Objective The aim of this study was to develop and conduct a preliminary clinical evaluation of SCAR molecular markers for identification of section Fumigati DNA, A. flavus , and A. niger . Methods The RAPD-PCR method was used to obtain specific bands from polymorphic patterns for the section Fumigati, A. flavus , and A. niger . Of the 34 polymorphic patterns obtained, only OPF-01, P54, and 1253 were selected, as they exhibited specific bands for the section Fumigati, A. flavus and A. niger , respectively. SCAR markers were generated from these bands by cloning and sequencing them. Subsequently, the specificity of the designed markers was evaluated by testing them against other species of the genus Aspergillus and other medically important genera. To evaluate sensitivity, serial dilutions (10 ng/µL– 0.001 ng/µL) of genomic DNA from the section Fumigati, A. flavus , and A. niger were used. Finally, the positivity of the SCAR-PCR assay was analyzed in galactomannan-positive bronchoalveolar lavage (BAL) samples from patients evaluated for aspergillosis. Results The primers OPF-01, P54, and 1253 produced unique bands for the section Fumigati, A. flavus , and A. niger , respectively. Subsequently, three primer pairs were designed: the first amplified a 180 bp band that showed section-level specificity for identifying the section Fumigati DNA; the second amplified a 193 bp band that recognized A. flavus ; and the third amplified a 220 bp band in A. niger and A. luchuensis . These SCAR markers were named AfOPF-01 180 , AfP54 193.1 , and An1253 220 . The limit of detection with the SCAR markers was 0.005 ng/µL. SCAR-PCR positivity of the marker OPF-01 180 in BAL samples was 19/51 (37.25%); SCAR-PCR positivity of the marker AfP54 193 . 1 was 3/51 (5.88%); There was no SCAR-PCR positivity for the marker An1253 220 . Conclusion Based on the results, the developed SCAR markers show preliminary clinical feasibility, identifying the section Fumigati DNA by the AfOPF-01 180 marker and at the species level by the AfP54 193.1 and An1253 220 markers. However, prospective, multicenter validation is required to evaluate its diagnostic clinical capability.