Abstract:
With increasing quantities of processed meat-based products being sold in New Zealand, it is important that their contents can be quickly and accurately identified. The ingredients listed on food products are not always accurate and the New Zealand Ministry of Health has identified risks associated with certain meat types, for example, bovine spongiform encephalopathy (BSE) is of particular concern. The identification of processed meat is made difficult by food processing techniques, which involve high temperature, humidity, salinity and pressure. All these conditions are capable of degrading DNA and other biological markers. In these circumstances, mitochondrial DNA (mtDNA) is an effective marker to target because it is present in high copy number. The entire mitochondrial genome sequence of most commercial species are well documented. The variable nature of the 16S rRNA gene makes it an ideal region to target for discriminating among species.
In this study a novel 16S rRNA multiplex PCR was designed and used to successfully discriminate among beef, chicken, pork and sheep species separately and as mixtures. The assay employs a 'universal' forward primer and 'coloured' species-specific reverse primers. The results showed that each of the four species could be detected in any combination, with any mixture of samples from the other three species, and no false positives or negatives were reported.
DNA degradation as a result of food processing was also investigated. Additional 16S rRNA primers were designed to investigate the effect of food processing on DNA fragmentation. Fragments were cloned and sequenced to determine the amount of base modifications, which may affect primer binding sites and hence detection of species. It was found that the major form of DNA degradation was strand breakage with very few base modifications.
This study presents a novel, sensitive species-specific 16S rRNA multiplex PCR assay, capable of identifying up to four species simultaneously for forensic science, and food authentication. This method can be easily integrated into a laboratory using existing fluorescently labelled fragment analysis methods.
