Desarrollo de métodos de RT-PCR en tiempo real para la cuantificación de mohos toxigénicos viables en alimentos

Résumé

Some mould species may grow and produce mycotoxins in foods including cereals and ripened meat products. Among them aflatoxins (AFs) and ochratoxin A (OTA) are the most important ones due to their toxicity and incidence. For this, it would be necessary to develop tools able to avoid or minimise mycotoxin accumulation in foods. In this sense, it would be used to have rapid and efficient methods such as RT-qPCR which are able to analyse changes in expression of genes involved in AFs and OTA biosynthesis before such mycotoxins are produced. These methods could be used in APPCC systems in the food industry. The main objective of this Doctoral Thesis was to develop RT-qPCR methods for quantification of viable moulds producers of AFs and OTA in foods. For this, a mould RNA extraction method directly from foods was optimised. This protocol consisted of grinding mycelium with the aid of a mortar and pestle and use of the commercial extraction kit RNeasy. Next, methods of RT-qPCR were designed and optimised on the basis of the aflP, aflR, aflS, otapks and otanps genes associated with AFs and OTA synthesis. Finally, the developed RT-qPCR methods were validated in food matrices where moulds normally grow. Such methods were able to detect expression of genes prior to mycotoxin production. Therefore, the methods of RT-qPCR optimized and developed in this Doctoral Thesis are useful and efficient tools which can be used to prevent mycotoxin accumulation in foods and, in this way, protect the consumer health.