On-chip PCR amplification of genomic and viral templates in unprocessed whole blood |
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Authors: | Dammika P Manage Yuen C Morrissey Alexander J Stickel Jana Lauzon Alexey Atrazhev Jason P Acker Linda M Pilarski |
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Affiliation: | (1) Department of Oncology, University of Alberta and Cross Cancer Institute, 11560 University Avenue, Edmonton, AB, T6G 1Z2, Canada;(2) Department of Laboratory Medicine and Pathology, University of Alberta, 8440-112 Street, Edmonton, AB, T6G 2B7, Canada;(3) Research and Development, Canadian Blood Services, 8249-114th Street, Edmonton, AB, T6G 2R8, Canada; |
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Abstract: | 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. |
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