PCR detection and identification of Alternaria species-groups in processed foods based on the genetic marker Alt a 1

In this study, a PCR method based on oligonucleotide primers targeting the Alt a 1 gene has been developed for the rapid detection of DNA from Alternaria spp. and for identification of Alternaria alternata, Alternaria porri, Alternaria radicina, and Alternaria infectoria species-groups in raw materials and processed food products. The assay design consists of two steps. First, a duplex PCR using primers Dir5cAlta1–Inv4Alta1 (that amplifies a specific DNA fragment of approximately 195 bp in all Alternaria spp.) and 18Sfweu–18Srveu (that amplifies a conserved 99 bp fragment on all the eukaryotic species), allows detection of Alternaria spp. DNA in foodstuffs with a high sensitivity and specificity. As a second step, identification of Alternaria species-groups is obtained through a seminested PCR method, without the need for sequencing PCR products. The specificity of the primer pairs designed was verified by PCR analysis of DNA from various Alternaria cultures, and also from several non target species. The detection limit of the method was approximately 102 CFU/ml, either in viable culture, heat inactivated culture or inoculated tomato pulp. Nevertheless, a sensitivity of 103 CFU/ml was obtained for tomato pulp inoculated with A. alternata or A. porri cultures heat inactivated at 90 °C for 5 min. PCR analysis of commercial foodstuff samples demonstrated the presence of DNA from A. alternata species-group in 100% of spoiled tomato samples, and 8% of tomato products, whilst 36.4% of cereal based infant food samples analyzed contained DNA from A. infectoria species-group.