Virtual Multiplexing Chamber-Based Digital PCR for Camel Milk Authentication Applications

In this work, we proposed a chamber-based digital PCR (cdPCR) microfluidic device that is compatible with fluorescence imaging systems for milk adulteration detection. The device enables the digitalization of PCR reagents, which are loaded into microchambers, and subsequent thermocycling for DNA amplification. Then, fluorescence images of the microchambers are captured and analyzed to obtain the total number of positive chambers, which is used to calculate the copy numbers of the target DNA, enabling accurate quantitative detections to determine intentional milk adulteration from accidental contaminations. The validation of this device is performed by camel milk authentication. We performed 25,600-chamber virtual multiplexing cdPCR tests using 40 × 40 chamber devices for the detection of DNA templates extracted from pure or mixed milk with different dilutions. Then, the cdPCR chip was used to authenticate blind milk samples, demonstrating its efficacy in real biotechnical applications.