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

Hezhi Hu,Xiaoshuai Meng,Chengzhuang Yu,Chunyang Wei,Shanshan Li,Junwei Li,Jingmeng Cheng

doi:10.3390/mi12060694

Hezhi Hu, Xiaoshuai Meng + Show 5 more

Open Access

https://doi.org/10.3390/mi12060694

Copy DOI

Abstract

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.

Highlights

The concentration of targets is important in the development of biosensors because in ultra-low concentration situations [1], the background may serve as a substrate noise.pre-concentration [2,3] or amplification [4] could provide solutions for biosensing.Among these approaches, polymerase chain reaction (PCR) has given access to a method of amplifying target deoxyribonucleic acid (DNA) across several orders of magnitude [5].Compared with real-time PCR, chamber-based digital polymerase chain reactionor droplet dPCR enable the absolute quantification [6,7] of nucleic acids without any calibrations
We present a 40 × 40 chamber-based digital PCR microfluidic device that is compatible with fluorescence image read-out systems and removes bubbles by a predegassing operation
These results demonstrate that the dPCR method is sensitive and specific for the rapid identification of chicken meat mixed in mutton

Summary

Introduction

The concentration of targets is important in the development of biosensors because in ultra-low concentration situations [1], the background may serve as a substrate noise.pre-concentration [2,3] or amplification [4] could provide solutions for biosensing.Among these approaches, polymerase chain reaction (PCR) has given access to a method of amplifying target deoxyribonucleic acid (DNA) across several orders of magnitude [5].Compared with real-time PCR, chamber-based digital polymerase chain reaction (dPCR)or droplet dPCR (ddPCR) enable the absolute quantification [6,7] of nucleic acids without any calibrations. Pre-concentration [2,3] or amplification [4] could provide solutions for biosensing. Among these approaches, polymerase chain reaction (PCR) has given access to a method of amplifying target deoxyribonucleic acid (DNA) across several orders of magnitude [5]. Compared with real-time PCR, chamber-based digital polymerase chain reaction (dPCR). Droplet dPCR (ddPCR) enable the absolute quantification [6,7] of nucleic acids without any calibrations. For microfluidic devices, digitalized micro chambers [8] or droplets [9] are usually used to perform a dPCR or ddPCR. The digitalized micro chamberbased dPCR features micro chamber arrays [10,11] containing a PCR mixture and target

Methods

Results

Discussion

Conclusion