Background and Objective
HIV viral load measurements play a critical role in monitoring disease progression in those who are on antiretroviral treatment. In order to obtain an accurate measurement, rapid sample preparation techniques are required. There is an unmet need for HIV extraction instruments in resource-limited settings, where HIV prevalence is high. Therefore, the objective of our study was to develop a three-dimensional (3D) microfluidic system to extract HIV-1 RNA with minimal electricity and without complex laboratory instruments.
A 3D microfluidic system was designed in which magnetic beads bound with nucleic acids move through immiscible oil–water interfaces to separate HIV-1 RNA from the sample. Polymerase chain reaction (PCR) amplification was used to quantify the total amount of HIV-1 RNA extracted as we optimized the system through chip design, bead type, carry-over volume, carrier RNA concentration, and elution buffer temperature. Additionally, the extraction efficiency of the 3D microfluidic system was evaluated by comparing with a Qiagen EZ1 Advanced XL instrument using 20 HIV-1-positive plasma samples.
Our method has near-perfect (100%) extraction efficiency in spiked serum samples with as little as 50 copies/mL starting sample. Furthermore, we report carry-over volumes of 0.31% ± 0.006% of total sample volume. Using the EZ1 Advanced XL as a gold standard, the average percentage HIV-1 RNA extracted using the microchip was observed to be 65.4% ± 24.6%.
From a clinical perspective, the success of our method opens up its possible use in diagnostic tests for HIV in the remote areas where access to vortexes and centrifuges is not available. Here we present a proof-of-concept device which, with further development, could be used for sample preparation at the point of care.
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Deng MY, Wang H, Ward GB, Beckham TR, McKenna TS. Comparison of six RNA extraction methods for the detection of classical swine fever virus by real-time and conventional reverse transcription-PCR. J Vet Diagn Investig. 2005;17:574–8.
Monleau M, Montavon C, Laurent C, Segondy M, Montes B, Delaporte E, et al. Evaluation of different RNA extraction methods and storage conditions of dried plasma or blood spots for human immunodeficiency virus type 1 RNA quantification and PCR amplification for drug resistance testing. J Clin Microbiol. 2009;47:1107–18.
Rahman MM, Elaissari A. Nucleic acid sample preparation for in vitro molecular diagnosis: from conventional techniques to biotechnology. Drug Discov Today. 2012;17:1199–207.
UNAIDS. How AIDS changed everything—MDG6: 15 years, 15 lessons of hope from the AIDS response. 2015. https://doi.org/10.1007/s13398-014-0173-7.2. http://www.unaids.org/en/resources/documents/2015/MDG6_15years-15lessonsfromtheAIDSresponse. Accessed 6 Mar 2019.
Bordelon H, Adams NM, Klemm AS, Russ PK, Williams JV, Talbot HK, et al. Development of a low-resource RNA extraction cassette based on surface tension valves. ACS Appl Mater Interfaces. 2011;3:2161–8.
den Dulk RC, Schmidt KA, Sabatté G, Liébana S, Prins MWJ. Magneto-capillary valve for integrated purification and enrichment of nucleic acids and proteins. Lab Chip. 2013;13:106–18.
Sur K, McFall SM, Yeh ET, Jangam SR, Hayden MA, Stroupe SD, et al. Immiscible phase nucleic acid purification eliminates PCR inhibitors with a single pass of paramagnetic particles through a hydrophobic liquid. J Mol Diagn. 2010;12:620–8.
Berry SM, Strotman LN, Kueck JD, Alarid ET, Beebe DJ. Purification of cell subpopulations via immiscible filtration assisted by surface tension (IFAST). Biomed Microdevices. 2011;13:1033–42.
Berry SM, Alarid ET, Beebe DJ. One-step purification of nucleic acid for gene expression analysis via immiscible filtration assisted by surface tension (IFAST). Lab Chip. 2011;11:1747–53.
Wang J, Morabito K, Erkers T, Tripathi A. Capture and separation of biomolecules using magnetic beads in a simple microfluidic channel without an external flow device. Analyst. 2013;138:6573–81.
Wang J, Morabito K, Tang JX, Tripathi A. Microfluidic platform for isolating nucleic acid targets using sequence specific hybridization. Biomicrofluidics. 2013;7(4):044107.
McCalla SE, Ong C, Sarma A, Opal SM, Artenstein AW, Tripathi A. A simple method for amplifying RNA targets (SMART). J Mol Diagn. 2012;14:328–35.
Wang J, Tai W, Angione SL, John AR, Opal SM, Artenstein AW, et al. Subtyping clinical specimens of influenza A virus by use of a simple method to amplify RNA targets. J Clin Microbiol. 2013;51:3324–30.
Cui FR, Wang J, Opal SM, Tripathi A. Isolating influenza RNA from clinical samples using microfluidic oil–water interfaces. PLoS One. 2016;11:e0149522.
Zhang L, Deraney RN, Tripathi A. Adsorption and isolation of nucleic acids on cellulose magnetic beads using a three-dimensional printed microfluidic chip. Biomicrofluidics. 2015;9(6):064118.
Lutfalla G, Uze G. Performing quantitative reverse-transcribed polymerase chain reaction experiments. Methods Enzymol. 2006;410:386–400.
Shaw KJ, Thain L, Docker PT, Dyer CE, Greenman J, Greenway GM, et al. The use of carrier RNA to enhance DNA extraction from microfluidic-based silica monoliths. Anal Chim Acta. 2009;652:231–3.
Butt AN, Swaminathan R. Overview of circulating nucleic acids in plasma/serum. Ann N Y Acad Sci. 2008;1137:236–42.
Troiano D, Deraney RN, Tripathi A. Effect of surfactants on carryover liquid volume in immiscible phase magnetic bead separation. Colloids Surf A Physicochem Eng Asp. 2016;513:188–95.
Berry SM, Lavanway AJ, Pezzi HM, Guckenberger DJ, Anderson MA, Loeb JM, et al. HIV viral RNA extraction in wax immiscible filtration assisted by surface tension (IFAST) devices. J Mol Diagn. 2014;16:297–304.
Britton S, Cheng Q, McCarthy JS. Novel molecular diagnostic tools for malaria elimination: a review of options from the point of view of high-throughput and applicability in resource limited settings. Malar J. 2016;15:88.
Sema M, Alemu A, Bayih AG, Getie S, Getnet G, Guelig D, et al. Evaluation of non-instrumented nucleic acid amplification by loop-mediated isothermal amplification (NINA-LAMP) for the diagnosis of malaria in Northwest Ethiopia. Malar J. 2015;14:44.
Tan SC, Yiap BC. DNA, RNA, and protein extraction: the past and the present. J Biomed Biotechnol. 2009;2009:574398.
The authors would like to thank Dr. Lei Zhang and Dr. Mark Bobrow for their guidance on this project.
Conflict of interest
Rachel N. Deraney, Derek Troiano, Richard Joseph, Soya S. Sam, Angela M. Caliendo, and Anubhav Tripathi declare no conflicts of interest relevant to this study.
This work was supported in part by the Providence/Boston Center for AIDS Research (P30AI042853) and in part by PerkinElmer.
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Deraney, R.N., Troiano, D., Joseph, R. et al. Vortex- and Centrifugation-Free Extraction of HIV-1 RNA. Mol Diagn Ther 23, 419–427 (2019). https://doi.org/10.1007/s40291-019-00394-1