Browsing by Author "Marta Tortajada"

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    Evaluation of DNA extraction methods for PCR detection of fungal and bacterial contamination in cocoa extracts
    (2009) Marta Tortajada; Pedro Vicente Martinez-Culebras; Veronica Navarro; Honorato Monzo Daniel Ramon
    Direct and sensitive PCR detection of contaminant microXora in cocoa extracts is aVected by the quality of the template DNA. This study compares the eYcacy of Wve diVerent commercial DNA extraction methods, selective enrichment broths and use of glycolitic enzymes to obtain quality DNA for PCR detection of both fungi and bacteria in artiWcially inoculated cocoa extract samples. PCR-based methods were applied to detect contaminant microXora in cocoa extracts using as model organisms: Aspergillus nidulans, Bacillus subtilis, Escherichia coli and Salmonella enterica. The quality of the extracted DNA was assessed in terms of PCR inhibitor content with results indicating that the HighPure PCR template (Roche) kit was the best methodology under the conditions assayed. PCR protocols using this commercial kit and a combination of glycolitic enzymes and enrichment procedures gave a detection limit of 100 conidia/g and 100 cfu/g for Wlamentous fungi and bacteria, respectively. The selected extraction and PCR procedures were also tested to assess their suitability for detecting Wlamentous fungi and bacteria on an industrial scale. They were sensitive enough to detect fungal and bacterial contaminants within the legally required limits. The results obtained with the molecular approach were in agreement with those of standard microbiological tests but require a considerably shorter analysis time. Thus, the molecular approach provides a sensitive and rapid alternative to check for microbial contamination in cocoa extracts.