Real-time PCR, and Recombinase Polymerase Amplification combined with SYBR Green I for naked-eye detection, along with Propidium Monoazide (PMA) for the detection of viable patulin-producing fungi in apples and by-products

Abstract

Nowadays, the application of rapid molecular-based methods such as PCR/qPCR and isothermal techniques like Recombinase Polymerase Amplification (RPA), has increased in order to overcome some of the drawbacks of traditional culture-based methods due to their high sensitivity and specificity. One of the main limitations of these techniques is their inability to differentiate between live and dead microorganisms. Patulin is a mycotoxin typically produced by fungi belonging to Penicillium spp. that exerts acute and chronic toxic effects to humans and animals. In the present study, one real-time PCR assay, and a RPA coupled with SYBR Green I for naked-eye detection, were developed for the detection of patulin-producing fungi. Primers and a probe were designed based on the idh gene of the patulin metabolic pathway. Furthermore, propidium monoazide was implemented in the DNA extraction protocol for the specific detection of viable fungi. The developed assays were able to detect down to 1.25 pg/μL (qPCR) and 23.8 pg/μL (RPA-SG) of pure P. expansum DNA. Finally, when artificially inoculated apples, and by-products, were analysed the LOD50 of the qPCR was found to be 8.1 × 103 spores/5 g of food sample. In the case of the RPA-SG, the determined LOD50 was 5.8 × 104 spores/5 g.