Development of rapid and sensitive loop-mediated isothermal method for on-site visual identification of tissue origin of pig by using mitochondrial COI gene sequences

Abstract

Context The loop-mediated isothermal amplification (LAMP) methods have great potential to identify the species origin of the tissue in meat and meat products at isothermal temperature and are also suitable for field conditions. Aim The present study aimed to develop a rapid, specific, and sensitive assay based on the LAMP technique for identification of tissue of pig origin. Methods The pig-specific primers were designed by targeting the mitochondrial COI gene. The amplification temperature and time for the LAMP reaction were optimised as 64°C and 45 min. The analysis of the amplified product was performed on the basis of the development of colour after the addition of intercalating SYBR Green I dye, and also by the ladder-like pattern on agarose-gel electrophoresis. Key results The assay was found to be highly specific for DNA templates of pig origin and showed no cross-reactivity with other food animals, viz. cattle, buffalo, sheep, and goats. The analytical sensitivities of the LAMP and PCR assays were recorded as up to 0.00001 ng and 0.1 ng respectively, of the absolute DNA content. The laboratory validation of the developed method was performed on blind samples and an admixture of meat from different food animals, viz. cattle, buffalo, sheep, goat and pig. The analysis could be performed in an hour by using supernatant from Phire Animal Tissue Direct PCR kit-treated tissue, excluding the complex process of nucleic acid extraction. Conclusion The LAMP assay was found to be cost-effective, easy to perform, and highly species-specific for pig tissue in meat and meat admixture. The result of the assay can be analysed with the naked eye without the need for sophisticated equipment. Compared with pre-standardised PCR assay, the developed LAMP method was quite sensitive and could be performed within 1 h, from sampling to analysis results. Implications The developed LAMP assay is low resource-based single-tube approach that could be exploited significantly in the fields of diagnostics, agriculture, and aquaculture.