Simple closed-system combining direct PCR and fluorescence for all types of milk and dairy product authentication

Abstract

Cow's milk is a popular food choice due to its nutritional value. However, some people have cow milk protein allergy. Alternative milk and dairy products are available, but they may be adulterated or fraudulently substituted with cow's milk to lower the production cost. Current conventional methods for cow DNA detection suffer from laboratory-based instruments and settings, which can be expensive, limit on-site usage, lack sensitivity, or cannot be used with highly degraded samples. Here, a simple closed-system based on direct PCR, which eliminates the need to extract DNA from samples, and SYBR Green I detection of PCR amplicons using the naked eye was developed and validated. Fluorescence detection was enhanced with the use of a self-designed portable LED lightbox, which mitigated color interference, enabling detection in products with diverse colorations without any sample preparation. Validation studies revealed 100% specificity when tested with 13 other species, over 99% accuracy with 208 cow’s milk and dairy products in both liquid and solid forms, 1×10−5 ng limit of detection, and 100% accuracy with blind testing. Market testing of 156 samples showed a fraud ratio of 0.0% in cow’s milk and dairy products, while non-cow’s milk and dairy products showed a fraud ratio of 10.7%, emphasizing the assay's capacity to identify fraudulent substitutions. Overall, this is the first time a cow-specific simple closed-system direct PCR with fluorescence has been used to authenticate fresh milk and dairy products, with one of the quickest turn-around time from sampling to detection of 50 min. It could be useful in a semi-laboratory setting or in the field with a portable PCR machine.