On-site paper-based Loop-Mediated Isothermal Amplification coupled Lateral Flow Assay for pig tissue identification targeting mitochondrial CO I gene

Abstract Identification of meat product adulteration is important to help fair-trade and to enable consumers to make informed choices. Detection of meat adulteration requires reliable analytical methods. Here, we report development of a loop-mediated isothermal amplification (LAMP) method for the detection of tissues of pig origin. We developed a kit containing lyophilized paper-buttons impregnated with the LAMP master-mix together with Lateral Flow Assay (LFA) paper strips. This paper-based LAMP-LFA kit is a ready to use device for remote settings and useful for the quick and efficient detection of tissue of pig origin. LAMP primers targeting mitochondrial COI gene and corresponding probes specific to LAMP amplicon were designed. LAMP reaction component, optimization details regarding thermal amplification protocol, hybridization conditions for probes, design of LFA paper-strips, and detection methods are provided. The paper-based LAMP-LFA platform allowed analysis of the amplified products using HNB dye-based quick visual method and gel electrophoresis-based validation to avoid personal bias. Assay limit-of-detection was 10 fg of DNA template in the reaction. Assay was validated on samples from different pigs, coded samples, binary meat admixture, diversely processed meat to mimic field situation, and supernatant from Phire Animal Tissue Direct PCR kit. The paper-based LAMP-LFA results were comparable with the pre-standardized species-specific polymerase chain reaction, a time consuming and expensive process. Lyophilized paper-based LAMP buttons in the form of a ready-to-use kit were developed to store reaction mixture. LAMP buttons were stable for up to four months when stored at 4 °C. Developed assay is capable of a rapid (3 hrs) identification of tissue of pig origin in tested samples, cost-effective, user-friendly, and requires minimal training making it a method of choice in remote point-of-care sites.