Development of a Protocol for Efficient DNA Extraction of Patulin-Producing Molds from Food for Sensitive Detection by PCR

Abstract

Early detection of patulin-producing molds in foodstuffs is very important for food industry and for ensuring the consumers’ health. For precise and sensitive detection of patulin-producing molds by polymerase chain reaction (PCR) protocol, a previously efficient DNA extraction from foods is necessary, since variations in the efficiency can affect the detectability. In this study, eight different DNA extraction methods including a wide variety of treatments were assayed and compared in order to select the most efficient. Methods which combined thermal conidia breakdown and DNA commercial extraction kits reached higher yields, obtaining more quantity and quality in DNA extraction than those methods without extraction kit. In the present work, a simple method for fungal DNA extraction which is relatively rapid by means of minimizing the use of costly chemicals such as sodium dodecyl sulfate, mercaptoethanol, etc., or dangerous such as phenol/chlorophorm, was developed. When the detection limits were evaluated after PCR amplification using DNA from nine inoculated food matrices with the eight methods tested, the method including thermal conidia breakdown, extraction with cetyl trimethylammonium bromide/phosphate-buffered saline (1:2) buffer, and E.Z.N.A. Fungal DNA Mini Kit was chosen as the most adequate for detecting patulin-producing molds by PCR. This method allowed detection limits ranged from 102–103 conidia/g.