Multiple authentications of high-value milk by centrifugal microfluidic chip-based real-time fluorescent LAMP

Abstract

Adulteration of food ingredients, particularly replacement of high-value milk with low-cost milk, affects food safety. For rapid and accurate identification of the possible adulterating milk species in an unknown sample, a centrifugal microfluidic chip-based real-time fluorescent multiplex loop-mediated isothermal amplification (LAMP) assay was developed to simultaneously detect milk from cow, camel, horse, goat, and yak. Using precoated primers in different reaction wells, the centrifugal microfluidic chip markedly simplified the detection process and reduced false-positive results. The entire amplification was completed within 90 min with a genomic detection limit of 0.05 ng/µL in cow, camel, horse, and goat milk and 0.005 ng/µL in yak milk. Using simulated adulterated samples for validation, the detection limit for adulterated milk samples was 2.5%, satisfying authentication requirements, as the proportion of adulterated milk higher than 10% affects economic interests. Therefore, this simple, centrifugal, microfluidic chip-based multiplex real-time fluorescent LAMP assay can simultaneously detect common milk species in commercial products to enable accurate labeling.