Colorimetric sensor for label free detection of porcine PCR product

Summary form only given. Selective detection of specific DNA sequences is increasingly getting momentum in clinical diagnosis, pathology, genetics and food analysis. The polymerase chain reaction (PCR) is a commonly used technique to amplify specific sequence segment in nearly all DNA-based assays. The use of PCR addresses both the sensitivity issues and sample purification steps producing a large quantity DNA from as little as single copy. However, post PCR analysis of PCR amplified DNA involves complex and time consuming electrophoresis, blot analysis or sequencing. In the current report, we prepared 40 ± 5 nm gold nanoparticles by citrate reduction of tetrachloroaurate and utilized them as colorimetric sensor for visual detection of a 17-bp sequence in 109 base-pair PCR products of porcine and deer mitochondrial genome amplified with a pair of common PCR primers. A 17-mer oligonucleotide, which was specific only for the porcine PCR product and polymorphic for other species, was mixed with both PCR products in separate tubes. The mixtures were denatured at 95 °C for 3 min and annealed at 51°C, 55°C and 59°C for 2 min. Addition of gold nanoparticle (AuNP) to porcine PCR product annealed at a critical temperature (55°C ) changed the color of colloidal particles from wine red to purple within 5 min. However, the wine red color of gold colloids was retained in deer PCR product. The assay was label-free, required no covalent modification of the DNA or AuNP-surfaces and took on the sensitivity of PCR. The result was determined visually and was strongly supported by absorption spectroscopy. We demonstrated the application of the assay in species identification for halal authentication and mismatch detection in genetic screening.