The limit of detection of PMA treatment associated with semi-nested touchdown PCR method in UHT milk was determined to be 102 CFU/mL of viable cells, which is lower than or similar to those described in other studies that have applied PMA-PCR assays to food analysis.
For example, a detection limit of 102 CFU/mL for Campylobacter jejuni (Josefsen et al., 2010) and Brochothrix thermosphacta (Mamlouk et al., 2012) was found in chicken carcass rinse and fresh salmon, respectively, while 103 CFU/g was described as the limit for Salmonella Typhimurium in lettuce (Liang et al., 2011). The difference in sensibility of the PCR method with or without the PMA pretreatment can result from the loss of cells during the additional PMA step and/or to the possible presence of B. sporothermodurans DNA in the late exponential phase cultures used to determine the PCR detection limit, possibly originating from cells that died during the bacterial growth phase. Additionally, the PMA-PCR method detection limit of 102 CFU/mL for B. sporothermodurans alone meets the criteria of the European Union (EU) and Brazilian legislation for the maximum count of mesophilic microorganisms in UHT milk
In conclusion, the new molecular assay developed here for the rapid, sensitive and specific detection of viable B. sporothermodurans cells could be a very useful tool for the early identification of undesirable B. sporothermodurans vegetative cells in milk, dairy products and additional food matrices. Thus, the application of this method could be of great value for the quality control of food products by monitoring the level of viable B. sporothermodurans during manufacture or storage and significantly reducing economic losses to the industry.