BARC/PUB/2023/0683

 
 

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

 
     
 
Author(s)

Dey, A.; Prakash, J.; Das, R.; Shelar, S.; Saini, A.; Cherian, S.; Patel, S. C.; Hassan, P. A.; Khandekar, A.; Dasgupta, K.; Misra, H. S.; Uppal, S.
(MBD;MG;MD;ChD)

Source

PNAS Nexus, 2023. Vol. 2 (3): Article no. pgad031

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 RDHLFA (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 (DNAB) specific to SARS-CoV-2 RNA (vRNA) to generate a vRNA-DNAB hybrid. The biotin-tagged vRNA-DNAB 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.

 
 
SIRD Digital E-Sangrahay