On-chip PCR amplification of genomic and viral templates in unprocessed whole blood

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 TM ). 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.

Molecular typing for blood group antigens within 40 min by direct polymerase chain reaction from plasma or serum

“…This approach has been established for some conventional agarose-based PCR (Awad & El-Tarras, 2011;Fuehrer et al, 2011) and in infectious disease testing (Fuehrer et al, 2011;Taylor et al, 2011). Direct PCR from unprocessed whole blood had also been combined with a rapid detection method by Manage et al (2010) utilizing a special microfluidic device. The discrimination of alleles remained a challenge in the presence of haemoglobin.…”

Polymeric-Based In Vitro Diagnostic Devices

“…Capture and purification of RNA using silica beads has been accomplished for E. coli RNA and viral RNA from influenza-A (H1N1)-infected mammalian cells [209] with real-time nucleic acid sequence-based amplification (NASBA) for target sequence amplification [210]. A combination of chemical and mechanical hybrid methods for bacterial cells lysis has also been reported [211].…”

Most probable number - loop mediated isothermal amplification (MPN-LAMP) for quantifying waterborne pathogens in < 25 min

“…Microfluidic polymerase chain reaction (PCR) systems are known to carry out DNA amplification directly from bacterial cells (Atrazhev et al, 2010; Manage et al, 2011) by using polymerases which are less prone to inhibition (Kermekchiev et al, 2009; Matheson et al, 2010; Zhang et al, 2010). Taq polymerase, the enzyme commonly used to amplify nucleic acids in PCR is inhibited by a variety of compounds such as hemoglobin, humic acid and cellular debris (Abu Al-Soud and Radstrom, 1998).…”