Development of a Panfungal Recombinase Polymerase Amplification (RPA) Method Coupled with Lateral Flow Strips for the Detection of Spoilage Fungi

Recombinase Polymerase Amplification combined with Lateral Flow (RPA-LF) detection was reported to be a good alternative to traditional culture-based methods, as well as other molecular techniques such as qPCR, PCR and Loop-Mediated Isothermal Amplification. The reasons for this are its simplicity, high sensitivity/specificity and ability for point of care (POC) applications. In this study, an RPA-LF assay was developed and evaluated for the detection of spoilage fungi in fruit-based products. In this sense, the universal primers ITS3 and ITS4 were labelled with digoxigenin and biotin, respectively. The method proved to be very sensitive with the ability to detect down to 1.2 pg/µL of pure fungal DNA. Furthermore, when the assay was applied in artificially contaminated samples, low detection limits were determined, in particular 1.0 CFU/50 g and 45.7 spores/50 g for yeasts and moulds, respectively. Overall, a rapid (24–48 h) and reliable assay was developed for the detection of fungi that could be applied for POC applications.