| Popis: |
The problem in meat adulteration and food fraud emphasised the requirement of developing accurate analytical approaches for the quantitative detection in helping the control of meat adulteration. In this study, the droplet digital Polymerase Chain Reaction (ddPCR) assays to quantify the ratios of pork DNA to the total amount of meat DNA were developed by challenging against DNA extracted from a range of gravimetrically prepared matrices of pork in beef. A single copy nuclear DNA gene, β-actin, was employed as a target gene, accompanied with myostatin gene as a cross species target for mammal and poultry meat background in order to quantifying approach. All the developed assays, singleplex, duplex and triplex did not show significant difference in quantification of pork content in beef background and demonstrated a good and comparable performance to the mass fractions. The singleplex assay provided more biases than the other two assays when performing with a low concentration of target species. The duplex assay provided a simultaneous quantification of pork and myostatin, whereas the triplex assay was able to detect pork, beef and myostatin with a decrease of technical error, cost and running time. All proposed methods allowed us to quantify pork addition in beef with a limit of quantification (LOQ) estimated at 0.1% (w/w) and a limit of detection (LOD) down to 0.01% (w/w). The developed triplex assay was also tested with commercial processed foods and showed the ability to determine not only the presence of particular pork or beef but also the quantitative purpose directly without standard curves. Hence, the developed ddPCR assays demonstrated a good trueness and precision of the methods in quantifying pork or beef content for meat adulteration. It is expected that these developed approaches can be applied to help regulators to confidently enforce food labelling obligations. |
