A novel One-pot rapid diagnostic technology for COVID-19

Abstract

Novel coronavirus disease (COVID-19) caused by SARS-CoV-2 is an ongoing global pandemic associated with high rates of morbidity and mortality. RT-qPCR has become the diagnostic standard for the testing of SARS-CoV-2 in most countries. COVID-19 diagnosis generally relies upon RT-qPCR-mediated identification of SARS-CoV-2 viral RNA, which is costly, labor-extensive, and requires specialized training and equipment. Herein, we established a novel one-tube rapid diagnostic approach based upon formamide and colorimetric RT-LAMP (One-Pot RT-LAMP) that can be used to diagnose COVID-19 without the extraction of specific viral RNA. The technique could visually detect SARS-CoV-2 within 45 minutes with a limit of detection of 5 copies per reaction in extracted RNA, and about 7.66 virus copies per μL in viral transport medium. The One-Pot RT-LAMP test showed a high specificity without cross-reactivity with 12 viruses including SARS-CoV, MERS-CoV, and human infectious influenza virus (H1N1/H3N2 of influenza A and B virus, ect. We validated this One-Pot RT-LAMP approach by its successful use for the analysis of 45 clinical nasopharyngeal swab samples, yielding results identical to those of traditional RT-qPCR analyses, while achieving good selectivity and sensitivity relative to a commercial RT-qPCR approach. As such, this One-Pot RT-LAMP technology may be a valid means of conducting high-sensitivity, low-cost and rapid SARS-CoV-2 identification without the extraction of viral RNA.

Two extraction-free reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2

“…Meanwhile, Li et al reported an extraction-free RT-LAMP method by incorporation of 6% formamide into reaction mixture. They managed to obtain 5 copies/μL within 45 min [ 15 ]. Wei et al reported a RT-LAMP based testing on RNA-spike samples by using an in-house lysis buffer.…”

A point-of-care SARS-CoV-2 test based on reverse transcription loop-mediated isothermal amplification without RNA extraction with diagnostic performance same as RT-PCR

“…The human internal control primer, RPP20, also performed relatively well on human gene dilutions. Of note, we used an inactivated whole virus rather than synthetic RNA transcripts as the standard control; hence a higher limit of detection (LOD) compared to other studies reporting similar performance to RT-PCR with lower LOD [ 36 ]. Zhou et al [ 37 ] recently reported that a whole virus quantified by RT-ddPCR would provide an ideal reference standard for quality control assessment from nucleic acid extraction to detection.…”

Coronavirus Disease 2019 (COVID‐19): Emerging detection technologies and auxiliary analysis

“…Based on conventional LAMP, researchers developed the capture and improved loop‐mediated isothermal amplification (Cap‐iLAMP) method, which combines a hybridization capture‐based RNA extraction of gargle lavage samples with an improved colorimetric RT‐LAMP assay and smartphone‐based color scoring. Cap‐iLAMP enables the detection of SARS‐CoV‐2‐positive samples in less than one hour 65,66 . Compared with RT‐PCR, this method amplifies DNA with high specificity, efficiency, and rapidity, without the need for expensive instruments, under isothermal conditions.…”

“…Cap‐iLAMP enables the detection of SARS‐CoV‐2‐positive samples in less than one hour. 65 , 66 Compared with RT‐PCR, this method amplifies DNA with high specificity, efficiency, and rapidity, without the need for expensive instruments, under isothermal conditions. These characteristics are a huge advantage for POCT.…”