An integrated digital polymerase chain reaction chip for multiplexed meat adulteration detection.

Meat adulteration detection is a common concern of consumers. Here, we proposed a multiplex digital polymerase chain reaction method and a low-cost device for meat adulteration detection. Using a polydimethylsiloxane microfluidic device, polymerase chain reaction reagents could be pump-free loaded into microchambers (40 × 40 chambers) automatically. Due to the independence of multiplex fluorescence channels, deoxyribonucleic acid templates extracted from different animal species could be distinguished by one test. In this paper, we designed primers and probes for four types of meat (beef, chicken, pork, and duck) and labeled each of the four fluorescent markers (hexachlorocyclohexane [HEX], 6-carboxyfluorescein [FAM], X-rhodamine [ROX], and cyanine dyes 5 [CY5]) on the probes. Specific detection and mixed detection experiments were performed on four types of meat, realizing a limit of detection of 3 copies/µL. A mixture of four different species can be detected by four independent fluorescence channels. The quantitative capability of this method is found to meet the requirements of meat adulteration detections. This method has great potential for point-of-care testing together with portable microscopy equipment.

Nucleic Acid Detection with Ion Concentration Polarization Microfluidic Chip for Reduced Cycle Numbers of Polymerase Chain Reaction.

Nucleic acid detection is widely used in disease diagnosis, food safety, environmental monitoring and many other research fields. The continuous development of rapid and sensitive new methods to detective nucleic acid is very important for practical application. In this study, we developed a rapid nucleic-acid detection method using polymerase chain reaction (PCR) combined with electrokinetic preconcentration based on ion concentration polarization (ICP). Using a Nafion film, the proposed ICP microfluidic chip is utilized to enrich the nucleic acid molecules amplified by PCR thermal cycles. To demonstrate the capability of the microfluidic device and the hybrid nucleic-acid detection method, we present an animal-derived component detection experiment for meat product identification applications. With the reduced cycle numbers of 24 cycles, the detection can be completed in about 35 min. The experimental results show that this work can provide a microfluidic device and straightforward method for rapid detection of nucleic acids with reduced cycle numbers.

Pre-Degassed Microfluidic Chamber-Based Digital PCR Device for Meat Authentication Applications.

Droplet digital polymerase chain reaction (ddPCR) suffers from the need for specific equipment and skilled personnel; thus, we here present a chamber-based digital PCR microfluidic device that is compatible with fluorescence image read-out systems and removes bubbles by a pre-degassed microfluidic device that consists of a pilot channel and micro chamber arrays. Digitalized PCR reagents are introduced into micro chambers, and thermocycles are taken to perform a DNA amplification process. Then, fluorescence images of a micro chamber array are read out and analyzed to obtain the total number of positive chambers. Thereby, the copy numbers of target DNA are calculated for quantitative detections. As a validation, this device is evaluated by the application of meat authentication. We performed dPCR tests using DNA templates extracted from a pure mutton DNA template with different dilutions. Then, the dPCR chip was used to identify the meat authentication using mutton-chicken mixtures with different mass ratios, showing its performance in real biotechnical applications.