Development of a rapid and ultra-sensitive RNA:DNA hybrid immunocapture based biosensor for visual detection of SARS-CoV-2 RNA

Anusree Dey; Jyoti Prakash; Rituparna Das; Sandeep Shelar; Ajay Saini; Susan Cherian; Sofia C Patel; Puthusserickal A Hassan; Ashwini Khandekar; Kinshuk Dasgupta; Hari Sharan Misra; Sheetal Uppal

doi:10.1093/pnasnexus/pgad031

Development of a rapid and ultra-sensitive RNA:DNA hybrid immunocapture based biosensor for visual detection of SARS-CoV-2 RNA

PNAS Nexus. 2023 Feb 1;2(3):pgad031. doi: 10.1093/pnasnexus/pgad031. eCollection 2023 Mar.

Authors

Anusree Dey^{1

2}, Jyoti Prakash^{3

2}, Rituparna Das^{1

2}, Sandeep Shelar⁴, Ajay Saini^{1

2}, Susan Cherian⁵, Sofia C Patel⁵, Puthusserickal A Hassan^{4

2}, Ashwini Khandekar⁵, Kinshuk Dasgupta^{3

2}, Hari Sharan Misra^{1

2}, Sheetal Uppal^{1

2}

Affiliations

¹ Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India.
² Homi Bhabha National Institute, Anushakti Nagar, Mumbai, 400094, India.
³ Materials Group, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India.
⁴ Chemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India.
⁵ Medical Division, Bhabha Atomic Research Centre, Anushakti Nagar, Mumbai, 400094, India.

Abstract

The Development of reliable and field-compatible detection methods is essential to monitoring and controlling the spread of any global pandemic. We herein report a novel anti-RNA:DNA hybrid (anti-RDH) antibody-based biosensor for visual, colorimetric lateral flow assay, using gold nanoparticles, coupled with transcription-mediated-isothermal-RNA-amplification (TMIRA) for specific and sensitive detection of viral RNA. We have demonstrated its utility for SARS-CoV-2 RNA detection. This technique, which we have named RDH-LFA (anti-RNA:DNA hybrid antibody-based lateral flow assay), exploits anti-RDH antibody for immunocapture of viral RNA hybridized with specific DNA probes in lateral flow assay. This method uses biotinylated-oligonucleotides (DNA_B) specific to SARS-CoV-2 RNA (vRNA) to generate a vRNA-DNA_B hybrid. The biotin-tagged vRNA-DNA_B hybrid molecules bind to streptavidin conjugated with gold nanoparticles. This hybrid complex is trapped by the anti-RDH antibody immobilized on the nitrocellulose membrane resulting in pink color signal leading to visual naked-eye detection in 1 minute. Combining RDH-LFA with isothermal RNA amplification (TMIRA) significantly improves the sensitivity (LOD:10 copies/µl) with a total turnaround time of an hour. More importantly, RDH-LFA coupled with the TMIRA method showed 96.6% sensitivity and 100% specificity for clinical samples when compared to a commercial gold standard reverse-transcription quantitative polymerase-chain-reaction assay. Thus, the present study reports a rapid, sensitive, specific, and simple method for visual detection of viral RNA, which can be used at the point-of-care without requiring sophisticated instrumentation.

Keywords: RNA:DNA hybrid; SARS-CoV 2; antibody; detection; lateral flow assay; transcription-mediated isothermal RNA amplification; viral; visual.