Vortex- and Centrifugation-Free Extraction of HIV-1 RNA
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ORIGINAL RESEARCH ARTICLE
Vortex‑ and Centrifugation‑Free Extraction of HIV‑1 RNA Rachel N. Deraney1 · Derek Troiano1 · Richard Joseph2 · Soya S. Sam3 · Angela M. Caliendo4 · Anubhav Tripathi1
© Springer Nature Switzerland AG 2019
Abstract 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. Methods 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. Results 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%. Conclusions 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.
Key Points A three-dimensional printed microchip was developed to extract HIV-1 RNA with only minimal electricity and no complex laboratory equipment. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s40291-019-00394-1) contains supplementary material, which is available to authorized users.
We present data on HIV-1 spiked serum samples, chip protocol optimization variables, as well as HIV-1-positive patient plasma samples.
* Anubhav Tripathi [email protected] 1
Center for Biomedical Engineering, School of Engineering, Brown University, 182 Hope Street, Providence, RI 02912, USA
2
PerkinElmer, 940 Winter Street, Waltham, MA 02451, USA
3
Division of Infectious Diseases, The Miriam Hospital, Providence, RI, USA
4
Department of Medicine, Warren Alpert Medical School of Brown University, Providence, RI, USA
1 Introduction Viral load testing is the cornerstone for monitoring HIVinfected pat
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