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Pork identification in raw meat using real-time polymerase chain reaction (PCR) was developed. Total DNA from meat samples were successfully extracted and found to be of high quality and produced clear PCR products. Porcine-specific molecular beacon probe and primers that amplifies 119 bp of the cytochrome b gene fragment of swine (Sus scrofa domestica) was used. Analysis of data showed that the C q (quantification cycle) from 10 ng/μl porcine DNA is (18.70 ± 0.12 to 19.08 ± 0.06). Meanwhile, the other samples exhibited negative result, which confirmed the specificity of the primers. The method also showed that the limit of detection of pork was 0.0001 ng. Based on the regression analysis of the standard curve, the 96% efficiency of real-time PCR was achieved with high correlation coefficient (r 2 = 0.9989). Sensitivity of the assay in discriminating pork as low as 0.1% (w/w) pork in pork–beef mixtures was also obtained. Reproducibility of the assay was successfully validated by applying sample and experimental replicates in every assay being conducted. Thus, this methodology could serve as a fast and sensitive method for detection of pork for meat species verification.

A novel class of nanobiosensor was developed by integrating a 27-nucleotideAluIfragment of swine cytochrome b (cytb) gene to a 3-nm diameter citrate-tannate coated gold nanoparticle (GNP). The biosensor detected 0.5% and 1% pork in raw and 2.5-h autoclaved pork-beef binary admixtures in a single step without any separation or washing. The hybridization kinetics of the hybrid sensor was studied with synthetic andAluIdigested real pork targets from moderate to extreme target concentrations and a sigmoidal relationship was found. Using the kinetic curve, a convenient method for quantifying and counting target DNA copy number was developed. The accuracy of the method was over 90% and 80% for raw and autoclaved pork-beef binary admixtures in the range of 5–100% pork adulteration. The biosensor probe identified a target DNA sequence that was several-folds shorter than a typical PCR-template. This offered the detection and quantitation of potential targets in highly processed or degraded samples where PCR amplification was not possible due to template crisis. The assay was a viable alternative approach of qPCR for detecting, quantifying and counting copy number of shorter size DNA sequences to address a wide ranging biological problem in food industry, diagnostic laboratories and forensic medicine.

We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620‐800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 ◦ C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork‐venison, pork‐shad and venison‐shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 μ gm l −1 swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species. (Some figures in this article are in colour only in the electronic version)

A 27-nucleotide AluI fragment of swine cytochrome b (cytb) gene was integrated to a 3-nm diameter citrate- tannate coated gold nanoparticle to fabricate a species specific nanobiosensor. The biosensor was applied to authenticate pork adulteration in autoclaved pork-beef mixtures. The sensor was found to be sensitive enough to detect 0.5 pork in raw and 2.5-h autoclaved mixed samples in a single step without any separation or washing. The hybridization kinetics of the hybrid sensor was studied with synthetic targets from moderate to extreme target concentrations and a sigmoidal relationship was found. The kinetic curve was used to develop a convenient method for quantifying and counting target DNA copy number. The biosensor probe was hybridized with a target DNA that was several-folds shorter than a typical PCR-template. This offered the detection and quantitation of potential targets in highly processed meat products or extensively degraded samples where PCR-based identification technique might not work due to the degradation of comparatively longer DNA. The assay was a viable alternative approach of qPCR for detecting, quantifying and counting copy number of shorter size DNA sequences in degraded samples to address a range of biological problems such as food analysis, biodiagnostics, environmental monitoring, genetic screening and forensic investigations.