Rapid and highly sensitive one-tube colorimetric RT-LAMP assay for visual detection of SARS-CoV-2 RNA

Abstract

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a highly contagious disease. To tame the continuously raging outbreak of COVID-19, developing a cheap, rapid and sensitive testing assay is absolutely imperative. Herein, we developed a one-tube colorimetric RT-LAMP assay for the visual detection of SARS-CoV-2 RNA. The assay integrates Si-OH magnetic beads (MBs)-based fast RNA extraction and rapid isothermal amplification in a single tube, thus bypassing the RNA elution step and directly amplifying on-beads RNA molecules with the visualized results. This one-tube assay has a limit of detection (LOD) as low as 200 copies/mL for sample input volumes of up to 600 μL, and can be performed in less than 1 hour from sample collection to result readout. This assay demonstrated an 100% concordance with the gold standard test RT-qPCR test by using 29 clinical specimens and showed high specificity. This one-tube colorimetric RT-LAMP assay can serve as an alternative platform for a rapid and sensitive diagnostic test for COVID-19 and is particularly suitable for use at community clinics or township hospitals.

Diagnostic assay and technology advancement for detecting SARS-CoV-2 infections causing the COVID-19 pandemic

“…Isothermal amplification techniques range in terms of total quantity of primers and enzymes employed, the temperature at which the amplification takes place, and the sorts of templates used. Table 2 [75][76][77][78][79][80][81][82][83][84][85][86][87] summarizes the several isothermal amplification platforms established for detecting SARS-CoV-2 in both laboratory and clinical samples.…”

“…Summary of the several isothermal amplification platforms established for SARS-CoV-2 diagnostics in both laboratory and clinical samples [Refs [75][76][77][78][79][80][81][82][83][84][85][86][87]. …”

Importance of sample input volume for accurate SARS-CoV-2 qPCR testing

“…50 μL of SARS-CoV-2 RNA was extracted from 200 μL of SARS-CoV-2 pseudovirus particles using a QIAamp Viral RNA Mini Kit (Qiagen, Dusseldorf, Germany) following the manufacture's instructions. While for SARS-CoV-2 pseudovirus particle samples with a range of input volumes from 100 to 1000 μL, 20 μL of viral RNA was purified and concentrated using a Si–OH magnetic bead-based viral RNA extraction method, which was developed by our lab [ 26 ]. In comparison with silica column-based nucleic acid extraction method, magnetic beads-based nucleic acid extraction protocol is much simpler, faster, and does not require the use of specialized equipment [ [27] , [28] , [29] ].…”

Challenges and Perspectives for Biosensing of Bioaerosol Containing Pathogenic Microorganisms

“…A thermoplasmonic-assisted dual-mode transducer was presented to detect the SARS-CoV-2 viruses in 30 min, where an amplification-free-based direct viral RNA detection and an amplification-based cyclic fluorescence probe cleavage detection were combined and demonstrated as a potential tool for fast clinical infection screening and real-time environmental monitoring [ 95 ]. Besides, some other molecular biological detection methods were also applied to detect the SARS-CoV-2 viruses [ 96 , 97 ]. Liu et al developed a 3D printed microfluidic chip to detect the SARS-CoV-2 viruses using a Flinders membrane for nucleic acid extraction, recombinase polymerase amplification, and loop-mediated isothermal amplification for sensitive detection, a smartphone-based platform for colorimetric signal monitoring.…”