A novel paper based loop mediated isothermal amplification and lateral flow assay (LAMP-LFA) for point-of-care detection of buffalo tissue origin in diverse foods

In this study, we have designed a paper based loop-mediated isothermal amplification-lateral flow (LAMP-LF) Assay that integrated the use of self-designed lyophilized paper LAMP buttons as ready-made LAMP master-mix for on-site detection of tissue of buffalo origin. Based on mitochondrial cytochrome b gene sequences, a pair of LAMP primers and probes were selected and designed, respectively. Herein, we optimized LAMP reaction components and thermal protocol to evaluate LAMP amplification in various paper matrices frequently used in diagnostics (cellulose, glass fiber, and nitrocellulose). The hybridization conditions of probes, as well as other components of paper LAMP-LF Assay, were further optimized. Our low-cost paper based system demonstrated high specificity and was able to detect as low as 10 fg of target DNA. The efficiency and accuracy of the assay were determined targeting coded meat/tissue samples, binary meat admixture, processed meat samples, Phire Animal Tissue Direct PCR kit, and by investigating the consistency between paper based LAMP-LFA and conventional PCR outcomes. Furthermore, the paper platform was allowed for refrigeration-temperature storage to determine its ability for deployment in low settings areas. In the current investigation, the time required from sample collection to detection was approximately 2-3 hr.