Dey Anusree, Prakash Jyoti, Das Rituparna, Shelar Sandeep, Saini Ajay, Cherian Susan, Patel Sofia C, Hassan Puthusserickal A, Khandekar Ashwini, Dasgupta Kinshuk, Misra Hari Sharan, Uppal Sheetal
Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India.
Homi Bhabha National Institute, Anushakti Nagar, Mumbai, 400094, India.
PNAS Nexus. 2023 Feb 1;2(3):pgad031. doi: 10.1093/pnasnexus/pgad031. eCollection 2023 Mar.
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) specific to SARS-CoV-2 RNA (vRNA) to generate a vRNA-DNA hybrid. The biotin-tagged vRNA-DNA 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.
开发可靠且适用于现场的检测方法对于监测和控制任何全球大流行疾病的传播至关重要。我们在此报告一种基于新型抗RNA:DNA杂交体（抗RDH）抗体的生物传感器，用于视觉比色侧向流动分析，该传感器使用金纳米颗粒，并结合转录介导的等温RNA扩增（TMIRA）来特异性灵敏地检测病毒RNA。我们已经证明了其在检测严重急性呼吸综合征冠状病毒2（SARS-CoV-2）RNA方面的效用。我们将这项技术命名为RDH-LFA（基于抗RNA:DNA杂交抗体的侧向流动分析），该技术利用抗RDH抗体在侧向流动分析中免疫捕获与特定DNA探针杂交的病毒RNA。此方法使用针对SARS-CoV-2 RNA（病毒RNA）的生物素化寡核苷酸（DNA）来生成病毒RNA-DNA杂交体。生物素标记的病毒RNA-DNA杂交分子与结合了金纳米颗粒的链霉亲和素结合。这种杂交复合物被固定在硝酸纤维素膜上的抗RDH抗体捕获，产生粉红色信号，可在1分钟内实现肉眼视觉检测。将RDH-LFA与等温RNA扩增（TMIRA）相结合可显著提高灵敏度（检测限：10拷贝/微升），总周转时间为1小时。更重要的是，与商业金标准逆转录定量聚合酶链反应检测相比，RDH-LFA与TMIRA方法相结合对临床样本的灵敏度为96.6%，特异性为100%。因此，本研究报告了一种快速、灵敏、特异且简单的病毒RNA视觉检测方法，该方法可在现场护理点使用，无需复杂仪器。
