An enhanced isothermal amplification assay for viral detection

Abstract

Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45 min from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.

Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2

“…2b, c). RNase H likely enhanced the sensitivity by increasing the efficiency of RT through degradation of DNA:RNA hybrid intermediates 15 .…”

“…Colorimetric LAMP assays enable high-throughput testing with minimal equipment requirements but often require purified samples to achieve high sensitivity. Improvements to RPA enable its use with unextracted samples and with increased sensitivity but are only compatible with lateral flow-based visual readouts, which can be tedious for larger sample numbers 15 . Although Abbott's ID NOW COVID-19 test using isothermal amplification with unextracted samples can report results in 5-13 min, this technology requires expensive equipment and has low throughput (~100 samples per machine per 24-h day) 16,17 .…”

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

“…Some RT-LAMP researches could rapidly detect SARS-CoV-2 with a low LOD (2 or 3 copies per reaction) [ 12 , 51 ], but require an RNA extraction step. Although some RT-LAMP methods have used unextracted swab samples to detect SARS-Cov-2 effectively, a pretreatment of samples lysed with a 95 °C lysis step in TCEP or VTM regent is required, and LOD of the assay is 50 copies per μL [ 35 , 36 ], which is higher than our work (5 copies per reaction in extracted RNA, and about 7.66 virus copies per μL in VTM); Other isothermal amplification technologies, such as improved RPA approach, report extraction-free SARS-CoV-2 with a low limit of detection (5 viral copies per reaction), however, it is necessary to manually insert the lateral flow strips into the open tube of each sample to visually interpret the results, which is tedious to achieve high throughput [ 21 ]. 2) One-tube reaction.…”

“…Isothermal amplification techniques, such as loop-mediated isothermal LAMP [ [12] , [13] , [14] ] and RPA [ 8 , 9 ], have become the methods of choice to detect SARS-CoV-2 without the need for complicated thermocyclers [ 19 , 20 ]. A high-sensitivity RPA method based on lateral flow strips may be cumbersome to achieve the high-throughput analysis of SARS-CoV-2 [ 21 ].…”

Extraction-free RT-LAMP to detect SARS-CoV-2 is less sensitive but highly specific compared to standard RT-PCR in 101 samples

“…The ID NOW compares favourably to GeneXpert [ 21 ], but also shares its disadvantages in terms of single sample per cartridge and single-source cartridges. Some kits, such as Twist Bioscience’s RPA-based assays [ 22 ] and the SHERLOCK LAMP/CRISPR assays, do not use special machines or cartridges, requiring only strip tubes or plates and p10 filter tips, hence can easily be scaled up to conduct hundreds or thousands of tests. New England Biolabs’ Colorimetric WarmStart LAMP kit can also be scaled and is available in Canada.…”