Desenvolvimento de PCR multiplex para detectar e identificar espécies de Aspergillus e Penicillium

Abstract

Aspergillus and Penicillium are the main fungal genera isolated from conditioned environments and are associated with adverse health effects of occupants of these places. Historically the fungal isolates are identified by macroscopic and microscopic analysis of colonies obtained by in vitro culture. These methods are falling into disuse, because they are time consuming and may present inaccurate results. In face these considerations new methods are required develop the fungal identification, which are faster, more specific and more sensitive than the others. That need motivated the purpose of this research: to develop a multiplex PCR protocol for identifying fungal species from mixed cultures. In this sense, a multiplex PCR was standardized with the ability to identify four species by reaction. The sample was composed by 183 fungal isolates of conditioned environments, obtained from the collection, which had their identification by conventional method (A. flavus n = 23, A. fumigatus n = 20, A. niger n = 50, A. ochraceus n = 20, P. citrinum n = 30, P. chrysogenum n = 20 and P. expansum n = 20) and multiplex PCR (A. flavus n = 14, A. fumigatus n = 20, A. niger n = 28, A. ochraceus n = 18, P. citrinum n = 12, P. chrysogenum n = 14 and P. expansum n = 7). To identify A. fumigatus and A. ochraceus the difference wasn’t statistically significant (p> 0.05), by the other side A. niger, P. citrinum and P. chrysogenum showed the greatest statistical differences when identification methods were compared. For the calculation of sensitivity and specificity, was chosen as the reference method the PCR. Except A. fumigatus, for all other species the conventional method was obtained the percentage of 100% sensitivity. Analyzing the specificity, A. niger, P. citrinum and P. expansum showed low percentage, respectively 74.12%, 82.18% and 87.91%. The identification by conventional method was more sensitive to species of Penicillium and greater specificity for species of Aspergillus. Of the 113 isolates identified by PCR, 61 (54%) were potential producers of mycotoxins by sorting Agar Coconut Milk, and accounted for 54.10% of potential producers ochratoxins, 29.5% and citrinin 16.4% of aflatoxin. In conclusion, the multiplex PCR capable of detecting four species by reaction proved to be a quick and easy to perform for the identification of fungal species from mixed culture resulting in air conditioned environments, besides having greater sensitivity and specificity than the conventional methods. Moreover, 54% of isolates identified by PCR showed up potential producers of mycotoxins by screening Agar Coconut Milk.