Development and validation of a real-time PCR assay specific for Clostridium estertheticum and C. estertheticum-like psychrotolerant bacteria.

A new real-time PCR assay was developed targeted to the psychrotolerant spoilage bacteria, Clostridum estertheticum, a causative agent of 'blown-pack' spoilage of vacuum packaged meats during chilled storage. Further, a robust validation of the sensitivity and specificity in different meat processing related matrices was carried out. Results show that real-time PCR is a valid method for the detection of C. estertheticum spores as long as consideration is given to the matrix being tested and the sensitivity of detection required. For meat, hide, blood/drip and environmental swabs it was possible to detect low numbers of C. estertheticum spores (approx 3 spores per ml) by direct real-time PCR (without pre-enrichment of the samples). For faeces and soil matrices, a cold temperature enrichment step was required prior to DNA extraction and real-time PCR analysis to increase the ability to detect samples containing C. estertheticum spores; this was particularly important when the samples contained low numbers of spores (less than 3 spores per ml). For matrices with high levels of PCR inhibitors such as soil, it was necessary to dilute the extracted DNA sample 100 fold especially for detection of high levels of contamination (greater than 10³ per ml) otherwise a pre-enrichment was required.