Point-of-care diagnostic methods for animal species determination are critical for rapid, simple, and accurate enforcement of food labelling. PCR is the most common method for species identification. However, the requirement of using a thermal cycler created drawbacks for the PCR application, particularly in low-resource settings. Hence, in this study, a method for porcine DNA detection using recombinase polymerase amplification (RPA), coupled with nucleic acid lateral flow immunoassay (NALFIA), was developed. Porcine-specific primers targeting pig (Sus scrofa) cytochrome b gene fragments specifically amplify a 197 bp fragment of the mitochondrial gene as being visualized by 2% agarose gel and PCRD NALFIA. The reaction temperature and time were 39 °C and 20 min, respectively. Herein, the specificity of the primers to porcine was confirmed after being assayed against six animal species, namely cow, goat, chicken, duck, dog, and rabbit. The porcine-specific RPA assay shows a high limit of detection of 0.01 ng/µL pork DNA. Based on the preliminary performance data obtained from this study, the potential of this method as a rapid and sensitive tool for porcine DNA detection in meat-based products is foreseen.

Real-time PCR was employed in this study to determine whether gelatine items from nearby marketplaces contained porcine DNA. The mitochondrial NADH dehydrogenase subunit 5 (MT-ND5) gene from pigs (Sus scrofa) was the subject of oligonucleotide primer design; the forward and the reverse primer sequences produced 161bp amplicon. SYBR Green fluorescent dye was used to detect swine DNA in samples of 22 different types of gelatine products, 12 of which had labels identifying their sources and indicating their certification as Halal, and the remaining 10 had neither labels nor certification information. The 40 cycles of positive amplication with a Ct value of 19.81 served as the criterion for determining the specificity of the primers. In this work, the detection threshold for porcine DNA is set at 0.001 µg/ml. The efficiency of the PCR in this study is 91.72%, and the R2 is 0.9985, which indicates good efficiency. The 12 labelled products exhibit positive amplifications for porcine gelatine products while exhibiting negative amplifications for both bovine and fish gelatine products in labelled samples. Nine out of the ten samples of gelatine products that were tested had positive amplifications without source or Halal labelling.