Development of a multiplex qPCR method for simultaneous quantification in dry-cured ham of an antifungal-peptide Penicillium chrysogenum strain used as protective culture and aflatoxin-producing moulds

A multiplex qPCR method to quantify the implantation of the antifungal-peptide-producing Penicillium chrysogenum RP42C and aflatoxin-producing moulds including a non-competitive IAC in dry-cured ham was developed. For this, the primer pairs F/R-Pc, F/R-omt and Tub-F1/R1 and the TaqMan probes, P-Pc, OMTprobe and Tubprobe targeting the pgafp, omt-1 and β-tubulin genes, respectively, were used. The ability of the optimized qPCR method to quantify simultaneously both non-toxigenic and toxigenic moulds in inoculated dry-cured ham was demonstrated since efficiency values ranged from 102.1 to 111.1%, and the limit of detection was between 2 and 3 log cfu/cm2 for both antifungal-peptide- and aflatoxin-producing moulds. In addition, suitability of multiplex qPCR to test implantation of protective P. chrysogenum as well as growth of aflatoxigenic strain in a controlled model system was carried out successfully. The multiplex qPCR allowed demonstrating that the protective strain of P. chrysogenum RP42C limits growth of the aflatoxin-producing Aspergillus flavus strain on dry-cured ham. These findings were confirmed by the results of aflatoxin analysis, since aflatoxin B1 was not detected when the protective P. chrysogenum RP42C was inoculated. Furthermore, the multiplex qPCR including an IAC quantified efficiently the implantation of protective culture P. chrysogenum RP42C in dry-cured ham after 6 months of incubation. Thus, the multiplex qPCR should be considered a sensitive and rapid tool to monitor the implantation of fungal protective cultures and to determine growth of aflatoxin-producing moulds in dry-cured ham as well.