Identificación y cuantificación de Clostridium tyrobutyricum por PCR a tiempo real en muestras de leche

Résumé

Detection and quantification of microorganisms by molecular techniques, particularly PCR, is being widely used in microbiological diagnosis. However, a method for the enumeration of the spoiling microorganism Clostridium tyrobutyricum is not currently available. This bacterium is the principal responsible for the late-blowing in hard and semi-hard cheeses. In this study, we have optimised a specific microbial DNA purification procedure from milk. We have also developed and assessed a realtime PCR assay, for the species-specific quantitative detection of C. tyrobutyricum. The PCR target was a region of the flagellin (fla) gene and the PCR system included an Internal Amplification Control (IAC). The co-amplification of the IAC in duplex PCR format enabled us to determine false negative results, quite common due to the high inhibitory effect of certain compounds of milk. The assay coprovedto be 100% specific, as determined with 19 Clostridium isolates and 40 non-Clostridium strains, and highly sensitive. Moreover, the correspondence between the numbers of clostridia estimated by the PCR system compared with the number of spores/mL calculated by the Most Probable Number (MPN) was evaluated.