Open AccessHIV detection from human serum with paper-based isotachophoretic RNA extraction and reverse transcription recombinase polymerase amplification
Author(s) -
Andrew T. Bender,
Benjamin Sullivan,
Jane Y. Zhang,
David Juergens,
Lorraine Lillis,
David S. Boyle,
Jonathan D. Posner
Publication year - 2021
Publication title -
analyst (london. 1877. online)/analyst
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.998
H-Index - 153
eISSN - 1364-5528
pISSN - 0003-2654
DOI - 10.1039/d0an02483j
Subject(s) - recombinase polymerase amplification , nucleic acid , reverse transcriptase , rna , rna extraction , rnase p , polymerase , lysis , bacteriophage ms2 , virology , biology , dna , polymerase chain reaction , microbiology and biotechnology , biochemistry , gene , coat protein
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 (ART). 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 (LMICs) 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 5000 HIV RNA copies per mL of serum. We then demonstrate detection of HIV virions and MS2 bacteriophage in human serum within 45-minutes.