Evaluation of methods for DNA extraction from Clostridium tyrobutyricum spores and its detection by qPCR.

Abstract Clostridium tyrobutyricum is the major agent that causes the blowing defect in cheese due to the germination of its dormant spores during the ripening stage. As a result, many of the affected cheeses show cavities and cracks, which cause the product loss in most cases. Nowadays, there is not a fast method capable of detecting milk contaminated with C. tyrobutyricum spores. The aim of this study has been to develop a fast and reliable method based on real time PCR (qPCR) to detect C. tyrobutyricum spores in raw milk. One of the main limitations has been to find a good procedure for the spore disruption to extract the DNA due to its high resistance. For this reason, different disruption methods have been tested, including chemical agents, bead beating, enzymatic and microwave treatment. Furthermore, an enzymatic treatment with subtilisin was applied for milk clarification and recovery of spores. The comparison of the assayed methods has been made using sterile milk spiked with C. tyrobutyricum spores, obtained in solid or liquid medium. The results showed that microwave treatment followed by a standard DNA purification step was found to be the best disruption method. The Ct values obtained for spores were higher than those found for vegetative cells by qPCR, for the same quantity of DNA. This difference could be due to the action of the Small Acid Soluble Proteins (SASP) in the DNA packaging of spores. Moreover, spores obtained in agar plate were found more resistant to disruption than those obtained in liquid medium. Subtilisin and microwave treatments were found to be successful for DNA extraction from C. tyrobutyricum spores in milk and subsequent identification by qPCR. However, the differences observed between the amplification of DNA from spores obtained in different media and from vegetative cells have to be taken into account to optimize a method for C. tyrobutyricum detection.