Development and evaluation of a real-time fluorescence, and naked-eye colorimetric, loop-mediated isothermal amplification-based method for the rapid detection of spoilage fungi in fruit preparations

Abstract

DNA-based techniques like PCR/qPCR have been applied for the detection of microorganisms in order to provide faster results, due to their high sensitivity and specificity, when compared to culture-based techniques. However, isothermal amplification techniques like Loop-mediated isothermal amplification (LAMP) have emerged lately allowing the simplification of the assays, and reduction of costs. The aim of this study was the development, and evaluation, of a panfungal LAMP assay (LAMP- 18S) for the fast detection of spoilage fungi in fruit preparations. In this sense, a set of primers was newly designed. Two different detection strategies were examined, namely real-time fluorescence and naked-eye colour detection. In addition, the results were compared against another published panfungal LAMP (LAMP-POW) and a qPCR assay, which served as the reference method. The developed method showed high sensitivity being able to detect down to 1.4 pg/reaction for yeasts and 170 pg/reaction for moulds. The addition of an enrichment step significantly reduced the LOD as the method could reliably detect yeasts with a LOD95 of 3.1 and 3.0 CFU/50 g for the fluorescent and colorimetric assay, respectively. The fluorescent assay showed “substantial agreement” with the reference method, while the colorimetric assay was “almost in complete concordance” with the reference method. Overall, a reliable and sensitive next-day detection method for fungi was achieved.