Journal Article

RNA-extraction-free nano-amplified colorimetric test for point-of-care clinical diagnosis of COVID-19

Alafeef, Maha, Moitra, Parikshit, Pan, Dipanjan, Dighe, Ketan

2021

Overview

The global pandemic of coronavirus disease 2019 (COVID-19) highlights the shortcomings of the current testing paradigm for viral disease diagnostics. Here, we report a stepwise protocol for an RNA-extraction-free nano-amplified colorimetric test for rapid and naked-eye molecular diagnosis of COVID-19. The test employs a unique dual-prong approach that integrates nucleic acid (NA) amplification and plasmonic sensing for point-of-care detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with a sample-to-assay response time of <1 h. The RNA-extraction-free nano-amplified colorimetric test utilizes plasmonic gold nanoparticles capped with antisense oligonucleotides (ASOs) as a colorimetric reporter to detect the amplified nucleic acid from the COVID-19 causative virus, SARS-CoV-2. The ASOs are specific for the SARS-CoV-2 N-gene, and binding of the ASOs to their target sequence results in the aggregation of the plasmonic gold nanoparticles. This highly specific agglomeration step leads to a change in the plasmonic response of the nanoparticles. Furthermore, when tested using clinical samples, the accuracy, sensitivity and specificity of the test were found to be >98.4%, >96.6% and 100%, respectively, with a detection limit of 10 copies/μL. The test can easily be adapted to diagnose other viral infections with a simple modification of the ASOs and primer sequences. It also provides a low-cost, rapid approach requiring minimal instrumentation that can be used as a screening tool for the diagnosis of COVID-19 at point-of-care settings in resource-poor situations. The colorimetric readout of the test can even be monitored using a handheld optical reader to obtain a quantitative response. Therefore, we anticipate that this protocol will be widely useful for the development of biosensors for the molecular diagnostics of COVID-19 and other infectious diseases. This protocol provides an RNA extraction–free nano-amplified colorimetric test that enables rapid detection of SARS-CoV-2 with the naked eye. The test uses plasmonic gold nanoparticles capped with antisense oligonucleotides as a colorimetric biosensor for point-of-care diagnosis of COVID-19.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/1647/1888/2005 / 631/1647/350/59 / 631/326/596/4130 / 639/301/357/354 / 639/925/352/1060 / Amplification / Analytical Chemistry / Antisense oligonucleotides / Biological Techniques / Biomedical and Life Sciences / Biosensors / Colorimetric analysis / Colorimetry / Colorimetry - instrumentation / Colorimetry - methods / Computational Biology/Bioinformatics / Coronaviruses / COVID-19 / COVID-19 - diagnosis / COVID-19 - virology / COVID-19 Nucleic Acid Testing - instrumentation / COVID-19 Nucleic Acid Testing - methods / Diagnosis / Gold / Gold - chemistry / Humans / Infectious diseases / Instrumentation / Life Sciences / Limit of Detection / Metal Nanoparticles - chemistry / Microarrays / Nanoparticles / Nucleic acids / Oligonucleotides / Oligonucleotides, Antisense - chemistry / Oligonucleotides, Antisense - genetics / Organic Chemistry / Pandemics / Plasmonics / Point of care testing / Protocol / Respiratory diseases / Response time / Ribonucleic acid / RNA / RNA, Viral - analysis / RNA, Viral - genetics / SARS-CoV-2 - genetics / SARS-CoV-2 - isolation & purification / Severe acute respiratory syndrome coronavirus 2 / Technology application / Viral diseases / Viral infections