ABSTRACT
A new lateral flow assay has been developed for meat authentication. The assay is based on gold nanoparticles and enables visual detection, by naked eye, of meat species-specific DNA sequences. The procedure includes DNA isolation from fresh meat samples, amplification of specific DNA sequences and detection by the lateral flow assay. The proposed assay is rapid and has been applied for the identification of four animal species: horse, pork, beef and sheep. The detection is completed within 25-30â¯min after amplification. The assay offers high detectability and good selectivity and reproducibility. As low as 0.01% of horse and 0.02% of pork DNA were detectable in binary mixtures by the reported lateral flow device.
Subject(s)
DNA/analysis , Food Analysis/methods , Meat/analysis , Metal Nanoparticles , Animals , Base Sequence , Cattle , Food Analysis/instrumentation , Gold , Horses/genetics , Metal Nanoparticles/chemistry , Red Meat/analysis , Reproducibility of Results , Sheep/genetics , Species SpecificityABSTRACT
A fluorometric lateral flow assay has been developed for the detection of nucleic acids. The fluorophores phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were used as labels, while a common digital camera and a colored vinyl-sheet, acting as a cut-off optical filter, are used for fluorescence imaging. After DNA amplification by polymerase chain reaction (PCR), the biotinylated PCR product is hybridized to its complementary probe that carries a poly(dA) tail at 3Î edge and then applied to the lateral flow strip. The hybrids are captured to the test zone of the strip by immobilized poly(dT) sequences and detected by streptavidin-fluorescein and streptavidin-phycoerythrin conjugates, through streptavidin-biotin interaction. The assay is widely applicable, simple, cost-effective, and offers a large multiplexing potential. Its performance is comparable to assays based on the use of streptavidin-gold nanoparticles conjugates. As low as 7.8 fmol of a ssDNA and 12.5 fmol of an amplified dsDNA target were detectable. Graphical abstract Schematic presentation of a fluorometric lateral flow assay based on fluorescein and phycoerythrin fluorescent labels for the detection of single-stranded (ssDNA) and double-stranded DNA (dsDNA) sequences and using a digital camera readout. SA: streptavidin, BSA: Bovine Serum Albumin, B: biotin, FITC: fluorescein isothiocyanate, PE: phycoerythrin, TZ: test zone, CZ: control zone.
