A Rapid Colorimetric and Fluorescent Assay of Aspergillus Fumigatus in Food by Loop-Mediated Isothermal Amplification.

Abstract

Aspergillus fumigatus is a foodborne mycete that can induce recurrent pneumonia, but the current detection methods have insufficient sensitivity and rapidity. Here, we aim to develop an efficient and sensitive loop-mediated isothermal amplification (LAMP) primer set for A. fumigatus detection. First, we designed a novel set of LAMP primers by targeting the Beta-tubulin (β-tub) gene. The LAMP reaction system was optimized by screening reaction temperature and betaine concentration. And then, the specificity of the proposed primers was verified by using 10 interferent microorganism species. The sensitivity of the designed method was compared with that of polymerase chain reaction (PCR) on pure cultures and complex matrix. The accuracy and response time of the method were examined by simulated samples. Our proposed primer set could accurately detect A. fumigatus from different food matrices with no response to other microorganisms. More intriguingly, this method possessed a low limit of detection (2 copies/reaction, 10-fold less than PCR), a short measuring time (<30 min), and a naked-eye readability. A real sample test demonstrates the good recovery rate and accuracy in apple, corn, milk, and other food matrix. Our proposed β-tub primer set provides great potential for rapid assessment of A. fumigatus contamination in food by integrating portable equipment and microscale reaction system.