Development of a molecular detection and differentiation system for ochratoxin A producing Penicillium species and its application to analyse the occurrence of Penicillium nordicum in cured meats

Abstract

A PCR method for differentiation and detection of the two known ochratoxin A producing Penicillium species, Penicillium verrucosum and Penicillium nordicum has been developed. It is based upon two genes of the ochratoxin A biosynthetic pathway, namely the ochratoxin A polyketide synthase gene ( otapksPN) and a non-ribosomal peptide syntethase gene ( otanpsPN) from P. nordicum. Both ochratoxin A producing Penicillia differ characteristically in the PCR result, making a taxonomic differentiation possible. P. verrucosum gives consistently only a positive reaction with the primers for the otanpsPN gene, whereas P. nordicum is positive for both genes. The PCR reaction is negative with all of other food related fungal species tested. This PCR system has been used to analyse 62 Penicillium strains isolated from cured meat products or ripening rooms, the natural habitat of P. nordicum. Among the 62 analysed strains 11 (18%) were positive with all specific PCR reactions. All 11 strains were able to produce ochratoxin A. In a RAPD analysis performed in parallel all 11 strains showed a pattern characteristic of P. nordicum, indicating the congruence of all data. None of the other strains isolated from cured meat produced ochratoxin A; most of them (30 out of 62) had a RAPD pattern characteristic for Penicillium nalgiovense. Interestingly some of the P. nalgiovense strains showed weak PCR product bands with varying length after electrophoresis. This was true for both primer pairs. None of these P. nalgiovense strains however produced detectable amounts of ochratoxin A. A more detailed analysis revealed that P. nalgiovense carries similar but non-transcribed sequences to the ochratoxin A biosynthetic genes of P. nordicum.