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

Abstract

The global pandemic of coronavirus disease 2019 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 nanoamplified 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.

A rapid RNA extraction-free lateral flow assay for molecular point-of-care detection of SARS-CoV-2 augmented by chemical probes

“…These ASOs are used to cap gold nanoparticles (AuNPs) which were then successfully used to selectively detect SARS-CoV-2 RNA by both photophysical ( Moitra et al, 2020 ) and electrochemical ( Alafeef et al, 2020a ) means. However, it was realized that only two of the ASOs among the four have optimum RNA binding capability and hence AuNPs conjugated with those two ASOs were used to develop a nano-amplified colorimetric test for COVID-19 diagnosis ( Alafeef et al, 2021 ). A comparative analysis of previously published COVID-19 detection technologies from our group is shown in Table S3 .…”

“…As a proof-of-concept experiment, genomic SARS-CoV-2 viral RNA was first reversed transcribed to cDNA and then amplified using suitably designed LAMP primers from a previous work ( Alafeef et al, 2021 ). The amplified viral DNA was then mixed with FAM and biotin labeled ASOs and allowed to incubate at 65 °C for 5 min.…”

Probing the mutation independent interaction of DNA probes with SARS-CoV-2 variants through a combination of surface-enhanced Raman scattering and machine learning

“…We propose to select four ASOs, two for the front (ASO1 and ASO2), and two targeting the end region (ASO3 and ASO4) of the gene. The ASOs will also target closely following sequences at each location ( Alafeef et al, 2020 , 2021a , 2021b , 2020 ; Dighe et al, 2021 ; Moitra et al, 2020 , 2021a , 2021b ). Accordingly, four ASOs were selected both for N and E genes, whereas only two were selected for the RdRp gene as ASOs with high enough binding energies are not available for this target ( Table 1 ).…”

Development of colorimetric sensors based on gold nanoparticles for SARS-CoV-2 RdRp, E and S genes detection

“…The selectivity and sensitivity of the sensors described were tested both qualitatively (visually) and quantitatively (absorbance measurements). It is worth mentioning that, compared to other approaches based on AuNPs’ aggregation [ [23] , [24] , [25] ], in this case, only one set of nanoparticles is needed. Those strategies require the precise equimolar combination of nanoparticles containing different oligonucleotides to target adjacent regions of the genomic nucleic acid.…”