A triple lateral flow strip assay based on multiplex polymerase chain reaction for simultaneous detection of chicken, pork and duck in adulterated meat

The increasing problem of meat adulteration significantly threatens consumer health and economic order. Therefore, developing an efficient and low-cost method for multi-species detection is essential to overcome the disadvantages of single-targeted, low-efficiency, and high-cost methods. This study presents a novel multiplex polymerase chain reaction (MPCR)-triple lateral flow strip (TLFS) integrated method, which enables the simultaneous, quantitative detection of chicken, duck, and pork ingredients in adulterated meat samples. Unlike traditional methods that target single species or require complex instrumentation, this method uniquely combines MPCR with TLFS to detect multiple species in one run, significantly reducing detection time and cost. This method uses MPCR to amplify genes specific to the three target types of meat and differentiate them by fluorophores (6-Fam, Cy5, and Digoxin). The TLFS consists of three separate lanes, each specific to one target meat amplicon (chicken, duck, or pork), allowing for the simultaneous detection of all three species from a single input sample. This setup enables the quantification of each species within a mixed meat sample by measuring the signal intensity from each lane, thus providing species-specific quantification in one run. MPCR amplicons are compatible with TLFS via antigen-antibody binding. By optimizing the reaction conditions, the method demonstrated good specificity, sensitivity, and stability. There were no cross-detections for three target meats (chicken, duck, and pork) and no false positives for seven others (horse, beef, lamb, camel, turkey, goose, and rabbit). The detection limit for chicken, duck, and pork species was low to 0.1 %, 0.5 %, and 0.05 % (wt%), respectively, which are all lower than the 1 % detection limit specified by the Chinese National Standard (GB/T 38164–2019). In the TLFS detection, meat samples can be qualified at 1 min and quantified after 7 min. The results of commercial samples showed that the method was consistent with the results of the national standard method, proving its reliability and practicality.