Rapid Detection of Porcine DNA in Meatball Using Recombinase Polymerase Amplification Couple with Lateral Flow Immunoassay for Halal Authentication

Abstract

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.

Amplification-Based CRISPR/Cas12a Biosensor Targeting the COX1 Gene for Specific Detection of Porcine DNA

“…The RPA-CRISPR/Cas12a fluorescence assay provides a significantly lower detection limit than previously reported isothermal amplification methods. 46 , 47 A similar detection limit has also been described in a qPCR. 48 When compared to other reported amplification-based CRISPR/Cas detection approaches, the proposed assay provides a notable detection limit ( Table S2 , Supporting Information).…”

“…The plot of the fluorescence intensities against the porcine DNA concentration displayed in Figure C exhibited a good linear correlation over the range of 5 to 1000 pg/μL with a coefficient of determination ( R 2 ) of 0.9921. The RPA-CRISPR/Cas12a fluorescence assay provides a significantly lower detection limit than previously reported isothermal amplification methods. , A similar detection limit has also been described in a qPCR . When compared to other reported amplification-based CRISPR/Cas detection approaches, the proposed assay provides a notable detection limit (Table S2, Supporting Information).…”

Molecular genetic methods for identifying raw materials in meat products: Diversity, opportunities and prospects

“…As a result of RPA, homogeneous dsDNA product of a given length is obtained. The successful application of RPA in combination with fluorescent and colorimetric detection [56,57] or with ICT [58][59][60][61][62][63][64][65] for detecting the impurities in meat (chicken, duck, pig, etc.) has been described.…”