Optimization of a real-time PCR assay for identification of Aspergillus fumigatus and Aspergillus niger from flour samples: Comparison of phenotypic and genotypic methods

Abstract

Aspergillus fumigatus and Aspergillus niger are airborne saprotrophs, which survive and grow on organic debris. It plays an essential role in food contaminations. Phenotypic methods for diagnosing this fungus are not very useful. Therefore, the purpose of this research was the optimization of the real-time PCR (RT-PCR) assay for the identification of Aspergillus fumigatus and Aspergillus niger in flour samples. In this study, 179 samples of flour were collected. Phenotypic methods were used for the initial separation of A. fumigatus. Then, to identify the A. fumigatus by molecular method, optimizing the RT-RCR assay and analysis of the melting curve were performed. Among 179 collected flour samples, 19 isolates (10.61%) of A. fumigatus and 16 isolates (8.93%) of A. niger were identified by the phenotypic method. Also, 11 isolates (6.14%) and nine isolates (5.02%) of A. fumigatus and A. niger were identified by RT-PCR method, respectively. Moreover, the phenotypic method's sensitivity and specificity for detecting A. fumigatus were 52.63%, 97.35%, and for A. niger was 66.73%, 96.95%. Besides, the RT-PCR method's sensitivity and specificity were determined by 100% to detect A. niger. However, the phenotypic method's low accuracy is the most critical factor in reporting false negatives and false positives. Also, due to the negative predictive value (NPV) and positive predictive value (PPV) of the RT-PCR assay, it is necessary to use this technique along with the phenotypic method to diagnose A. fumigatus and A. niger.