Paper-based loop-mediated isothermal amplification and lateral flow (LAMP-LF) assay for identification of tissues of cattle origin

Abstract

The present study was made with the objectives of development and standardization of cattle specific paper-based loop-mediated isothermal amplification cum lateral flow assay (LAMP-LFA), as a Point-of-care test (POCT) for identification of tissue of cattle origin. The components of standardized LAMP reaction utilizing cattle specific primer sets were lyophilized over paper buttons, identified best as the carrier of LAMP reagents. Based on probable LAMP amplicon, a pair of probes was designed, tagged and its hybridization with the amplified product of paper LAMP reaction was optimized. The components of lateral flow assay for detection of probe hybridized LAMP products were standardized. Analysis of successful amplification was made by using HNB dye, LAMP-LFA strip, and also by the typical ladder-like pattern on gel electrophoresis. The assay was found highly specific for cattle with an analytical sensitivity of 0.1 pg of absolute DNA. Laboratory validation carried out on samples from different individuals of cattle, coded samples, binary meat admixture, and heat-processed cattle tissues substantiated the accuracy of the assay. Comparison with pre-standardized species-specific PCR assay taken as gold standards revealed 100% conformity. The field utility of the developed assay was further established by its compatibility with the commercial kit eliminating the lengthy DNA extraction step and storage stability of LAMP reagent carrier buttons for 4 months under refrigeration. Thus, the developed assay capable of the result within 3 h in resource-limited settings can be used as POCT for identification of tissue of cattle origin.

Keywords: HNB dye; Lyophilized; Meat speciation; Paper based l loop mediated isothermal amplification-lateral flow (LAMP-LF) assay; Point-of-care test (POCT).