Integrated Capillary Electrophoresis Microsystem for Multiplex Analysis of Human Respiratory Viruses

摘要

We developed a two-layer, four-channel polymerase chainnreaction (PCR)-capillary electrophoresis microdevice thatnintegrates nucleic acid amplification, sample cleanup andnconcentration, capillary electrophoretic separation, andndetection for multiplex analysis of four human respiratorynviral pathogens, influenza A, influenza B, coronavirusnOC43, and human metapneumovirus. Biotinylated andnfluorescently labeled double-stranded (ds) deoxyribonucleicnacid (DNA) amplification products are generatednin a 100 nL PCR reactor incorporating an integratednheater and a temperature sensor. After amplification, thenproducts are captured and concentrated in a cross-linkednacrylamide gel capture matrix copolymerized with acrydite-nfunctionalized streptavidin-capture agents. Thermalndehybridization releases the fluorescently labeled DNAnstrand for capillary electrophoresis injection, separation,nand detection. Using plasmid standards containing thenviral genes of interest, each target can be detected startingnfrom as few as 10 copies/reactor. When a two-step reversentranscription PCR amplification is employed, the devicencan detect ribonucleic acid (RNA) analogues of all fournviral targets with detection limits in the range of 25-100ncopies/reactor. The utility of the microdevice for analyzingnsamples from nasopharyngeal swabs is demonstrated.nWhen size-based separation is combined with four-colorndetection, this platform provides excellent product discrimination,nmaking it readily extendable to higher-ordernmultiplex assays. This portable microsystem is also suitablenfor performing automated assays in point-of-carendiagnostic applications.