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HIV Detection from Human Serum with Paper-Based Isotachophoretic RNA Extraction and Reverse Transcription Recombinase Polymerase Amplification

preprint
submitted on 08.01.2021, 00:35 and posted on 11.01.2021, 05:08 by Andrew Bender, Benjamin Sullivan, Jane Zhang, David Juergens, Lorraine Lillis, David Boyle, Jonathan D. Posner

The number of people living with HIV continues to increase with the current total near 38 million, of which about 26 million are receiving antiretroviral therapy. These treatment regimens are highly effective when properly managed, requiring routine viral load monitoring to assess successful viral suppression. Efforts to expand access by decentralizing HIV nucleic acid testing in low- and middle-income countries has been hampered by the cost and complexity of current tests. Sample preparation of blood samples has traditionally relied on cumbersome RNA extraction methods, and it continues to be a key bottleneck for developing low-cost POC nucleic acid tests. We present a microfluidic paper-based analytical device (µPAD) for extracting RNA and detecting HIV in serum, leveraging low-cost materials, simple buffers, and an electric field. We detect HIV virions and MS2 bacteriophage internal control in human serum using a novel lysis and RNase inactivation method, paper-based isotachophoresis (ITP) for RNA extraction, and duplexed reverse transcription recombinase polymerase amplification (RT-RPA) for nucleic acid amplification. We design a specialized ITP system to extract and concentrate RNA, while excluding harsh reagents used for lysis and RNase inactivation. We found the ITP µPAD can extract and purify 5,000 HIV RNA copies per mL of serum. We then demonstrate detection of HIV virions and MS2 bacteriophage in human serum within 45-minutes.

Funding

Electrokinetic paper diagnostic platform: 15-minute, quantitative nucleic acid amplification for viral pathogens in whole blood

National Institute of Biomedical Imaging and Bioengineering

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History

Email Address of Submitting Author

bendera@uw.edu

Institution

University of Washington

Country

United States

ORCID For Submitting Author

0000-0003-4349-4488

Declaration of Conflict of Interest

We declare no conflict of interest.

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