On-chip PCR amplification of genomic and viral templates in unprocessed whole blood
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Performing medical diagnosis in microfluidic devices could scale down laboratory functions and reduce the cost for accessible healthcare. The ultimate goal of such devices is to receive a sample of blood, perform genetic amplification (polymerase chain reaction—PCR) and subsequently analyse the amplified products. DNA amplification is generally performed with DNA purified from blood, thus requiring on-chip implementation of DNA extraction steps with consequent increases in the complexity and cost of chip fabrication. Here, we demonstrate the use of unprocessed whole blood as a source of template for genomic or viral targets (human platelet antigen 1 (HPA1), fibroblast growth factor receptor 2 (FGFR2) and BK virus (BKV)) amplified by PCR on a three-layer microfluidic chip that uses a flexible membrane for pumping and valving. The method depends upon the use of a modified DNA polymerase (Phusion™). The volume of the whole blood used in microchip PCR chamber is 30 nl containing less than 1 ng of genomic DNA. For BKV on-chip whole blood PCR, about 3000 copies of BKV DNA were present in the chamber. The DNA detection method, laser-induced fluorescence, used in this article so far is not quantitative but rather qualitative providing a yes/no answer. The ability to perform clinical testing using whole blood, thereby eliminating the need for DNA extraction or sample preparation prior to PCR, will facilitate the development of microfluidic devices for inexpensive and faster clinical diagnostics.
KeywordsOn-chip PCR Microfluidics Whole blood PCR Phusion Taq
This work was supported by an Interdisciplinary Team Grant from the Alberta Heritage Foundation for Medical Research (AHFMR). LMP is the Canada Research Chair in Biomedical Nanotechnology and this work was funded in part by the Chair’s program. We thank Dr. Xiao-Li Pang, Provincial Laboratories for Public Health, Edmonton, Alberta for providing BKV DNA and Paul R. Dumais for the fabrication of the chips.
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