Combination of Loop-Mediated Isothermal Amplification and AuNP-Oligoprobe Colourimetric Assay for Pork Authentication in Processed Meat Products

Pork adulteration is a major concern for Muslims and Jews whose diets are restricted by religious beliefs, as well as those who are allergic to pork meat and its derivatives. Accurate pork authentication is of great importance to assist this demographic group of people in making decision on their product purchase. The aim of this study was to develop a new analytical method for pork authentication in processed meat products based on a combination of loop-mediated isothermal amplification (LAMP) and AuNP-nanoprobe colourimetric assay. The LAMP conditions were first optimised to obtain the highest yield of amplified DNA products within the shortest time. Oligoprobe-functionalised AuNPs were then hybridised with LAMP-DNA amplicons and subsequently challenged with MgSO4 at a high concentration to induce AuNP aggregation. In the presence of pork DNA, the colloidal AuNP-probe remained unchanged in its red colour, which indicates the dispersion of AuNPs. In contrast, in the absence of pork DNA, the colour was changed to colourless as a result from the aggregation of AuNPs. The LAMP-AuNP-nanoprobe assay offers a high sensitivity with a limit of detection as low as 100 pg of pork DNA. The assay is highly specific to pork content without cross-reactivity with the other meat species tested. The assay developed herein can become a simple, inexpensive, precise, and rapid analytical tool for small laboratories or the general public interested in halal food authentication

Sensitive Detection of Chicken Meat in Commercial Processed Food Products Based on One-Step Colourimetric Loop-Mediated Isothermal Amplification

Meat adulteration has been a great concern in the food industry worldwide. Due to its lower cost, chicken is generally used as an adulterant in several meat products. The main objective of this research was to develop a novel detection platform based on colourimetric loop-mediated isothermal amplification (LAMP) for authenticating chicken content in raw and processed meat products. Conditions for the colourimetric LAMP were thus optimised. Neutral red, a pH-sensitive indicator, was introduced into the LAMP reactions to help distinguish the positive/negative outcomes. LAMP reaction containing amplified LAMP amplicons changed its colour from yellow to pink/magenta, while the reaction without amplified DNA products remained in its original yellow colour. This single-step colourimetric LAMP was also validated for its high specificity towards chicken DNA without any cross-reactivity with the DNA from other types of meat. The limits of detection (LOD) for chicken DNA and chicken in binary meat admixtures were 1 pg and 0.01% (w/w), respectively. From 33 different commercial processed food samples, the assay confirmed chicken content in 14 declared chicken-containing products and in 8 products without the declaration of chicken content, while the remaining 11 meat samples without the declaration of chicken content showed no detectable chicken DNA. Furthermore, without the requirement of DNA purification, the direct colourimetric LAMP assay is capable of accurate authentication of chicken content in raw meat matrices and commercial processed food samples, making it a valuable analytical tool to facilitate on-site food authentication, as well as in low-resource laboratory settings

Accurate determination of meat mass fractions using DNA measurements for quantifying meat adulteration by digital PCR

The alarming problem of meat adulteration emphasises the demand for accessible analytical approaches for food regulatory agencies to detect and, specially, to measure altered meat fractions. This study proposes a novel cross-species triplex droplet digital polymerase chain reaction (ddPCR) assay to simultaneously identify and quantify the ratios of pork/beef meat fractions from a total DNA content, including processed and autoclaved meat, without requiring a standard, achieving high sensitivity with a limit of quantification estimated at 0.1% (w/w) and a limit of detection down to 0.01% (w/w). A single copy nuclear gene, β-actin, was employed as a target, accompanied with myostatin gene as a cross-species target to quantify the meat background. The duplex assay provided a simultaneous quantification of pork and myostatin, whereas the triplex assay was able to detect pork, beef and myostatin with a decrease of technical error, cost and time

Affimer-based impedimetric biosensors for fibroblast growth factor receptor 3 (FGFR3): a novel tool for detection and surveillance of recurrent bladder cancer

Fibroblast growth factor receptor 3 (FGFR3) is a transmembrane tyrosine kinase protein in the fibroblast growth factor receptor family, which plays a key role in many biological processes. Over-expression and activating mutations in FGFR3 are frequent in non-invasive bladder cancer, highlighting this protein as a potential biomarker for recurrent bladder cancer detection. Affimer reagents isolated against recombinant FGFR3 were assessed for their affinity using double-sandwich ELISA and SPR. Anti-FGFR3-14 and FGFR3-21 Affimer proteins showed strong binding to FGFR3 and were used for fabrication of impedimetric electrochemical biosensors. A decrease in impedance was observed when the sensors were exposed to increasing concentrations of FGFR3. The successful impedimetric biosensors were capable of detecting sub-pM to nM concentrations of recombinant FGFR3 protein in phosphate buffered saline as well as in synthetic urine