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.

Pork adulteration is a major concern for Muslims and Jews whose diets are restricted by religious beliefs, as well as those who are allergic to pork meat and its derivatives. Accurate pork authentication is of great importance to assist this demographic group of people in making decision on their product purchase. The aim of this study was to develop a new analytical method for pork authentication in processed-meat products based on a combination of loop-mediated isothermal amplification (LAMP) and AuNP-nanoprobe colourimetric assay. The LAMP conditions were first optimised to obtain the highest yield of amplified DNA products within the shortest time. Oligoprobe-functionalised AuNPs were then hybridised with LAMP-DNA amplicons, and subsequently challenged with MgSO ₄ at a high concentration to induce AuNP aggregation. In the presence of pork DNA, the colloidal AuNPs-probe remained unchanged in its red colour, which indicates the dispersion of AuNPs. In contrast, in the absence of pork DNA, the colour was changed to colourless as a result from the aggregation of AuNPs. The LAMP-AuNP-nanoprobe assay offers a high sensitivity with a limit of detection as low as 100 pg of pork DNA. The assay is highly specific to pork content without cross-reactivity with the other meat species tested. The assay developed herein can become a simple, inexpensive, precise, and rapid analytical tool for small laboratories or the general public interested in halal food authentication.

Meat adulteration, particularly the substitution of duck with chicken, threatens food authenticity, consumer trust, and regulatory compliance. However, converting DNA copy number measurements into meat mass fractions remains challenging. This study developed a digital PCR (dPCR)-based quantitative for determining chicken in duck. Singleplex and duplex dPCR assays targeting single-copy nuclear genes specific to chicken, duck and the conserved myostatin gene were developed and optimized. Five sets of binary chicken-in-duck mixtures were prepared and analyzed across 0–100% (w/w) to account for biological variability among meat sources. Five conversion approaches were evaluated: two conversion factor approaches based on the chicken-to-duck (k_cd) and chicken-to-myo (k_myo) copy number ratios, two corresponding approaches without conversion factors (No_kcd and Myo), and a linear regression model (Std). Quantitative performance of all five conversion approaches was concentration-dependent, and no single approach was optimal across the entire range. Conversion factor approaches generally improved agreement with gravimetrically assigned values at low to intermediate levels but overestimated higher chicken fractions. The Std approach performed well at ≥5% (w/w) but was less reliable at lower levels. Approaches selected based on their quantitative performance were further evaluated across different sample compositions and processing conditions and applied to commercial duck products. Overall, dPCR combined with an appropriate conversion approach may support quantitative determination of chicken in duck meat for food authenticity testing.