Rapid and Visual Detection of SARS-CoV-2 RNA Based on Reverse Transcription-Recombinase Polymerase Amplification with Closed Vertical Flow Visualization Strip Assay

Abstract

The global COVID-19 epidemic, ongoing since the initial outbreak in 2019, has caused panic and huge economic losses worldwide. Due to the continuous emergence of new variants, COVID-19 has been responsible for a higher proportion of asymptomatic patients than the previously identified SARS and MERS, which makes early diagnosis and prevention more difficult.

Detection of SARS-CoV-2 Based on Nucleic Acid Amplification Tests (NAATs) and Its Integration into Nanomedicine and Microfluidic Devices as Point-of-Care Testing (POCT)

“…Sample preparation must be minimal to avoid RNA extraction, which is a time-consuming step and affects sensitivity, depending on the extraction method [ 122 ]. Many of the approaches we have reviewed have taken this into account and can be performed rapidly, thanks to isothermal amplification kinetics and the tolerance of inhibitors, or the specificity of functionalized nanoparticles, such as glass slides or aptamers [ 53 , 66 , 104 , 105 , 108 , 109 , 112 ].…”

“…It could be coupled to a lateral flow assay (LFA) like other NAATs and provide visual readouts as a qualitative method, or be automated in microdevices or point-of-care biosensors. Some strategies will be reviewed in the next sections, but examples of the approaches that include RPA with CRISPR-based methods are SHERLOCK, with advances as presented by Song et al (2023) [ 53 ], NanoPEIAs, etc. [ 54 , 55 ].…”

“…RT-RPA has also been combined with lateral flow in a highly sensitive assay which is able to detect as little as one copy of each variant, with no cross-reactions with other respiratory viruses, and has a performance time of 25 min, with visual readouts [ 53 ].…”

Recent Uses of Paper Microfluidics in Isothermal Nucleic Acid Amplification Tests

“…Another way to compare amplification methods is by analytical sensitivity or limit of detection (LOD), which is also challenging, as the LOD significantly varies according to the target and detection method. For example, when detecting SARS-CoV-2 with paper microfluidic isothermal amplification tests, one colorimetric detection assay with RPA yielded a LOD of 20 copies/μL, while another colorimetric RPA assay reported LODs of 1 and 0.77 copies/μL for the delta and omicron variants, respectively [ 38 , 40 ]. This result suggests a wide variation in analytical sensitivity or LOD within the same amplification method.…”

“…For LFIA detection, probes and/or primers are tagged, generating tagged amplicons. These tagged amplicons are loaded onto the strip with a bioreceptor (e.g., antibodies) conjugated with a colorimetric indicator (e.g., gold nanoparticles = AuNPs) to detect a range of targets such as parasites, bacteria, viruses, genetic modification in crops, and even antibiotics [ 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 ]. The solution is migrated spontaneously via capillary action, and the tagged amplicons + bioreceptor + colorimetric indicator complexes are captured and accumulated on the test line of a strip, where the bioreceptor is immobilized.…”