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

Maha Alafeef, Parikshit Moitra, Ketan Dighe, Dipanjan Pan

Research output: Contribution to journalReview articlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3141-3162
Number of pages22
JournalNature Protocols
Volume16
Issue number6
Early online dateApr 30 2021
DOIs
StatePublished - Jun 2021

Keywords

  • COVID-19
  • severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
  • Novel coronavirus
  • Pandemic
  • 2019-nCoV
  • Coronavirus

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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