An enhanced isothermal amplification assay for viral detection

Jason Qian,Michael Springer,Sarah A Boswell,Benjamin L Gaudio,Rebecca H Ward,Zhi-Xiang Lu,Jesse M Fajnzylber,Mary E Pettit,Jonathan Z Li,Christopher Chidley,Ryan T Ingram

doi:10.1038/s41467-020-19258-y

Jason Qian, Michael Springer + Show 9 more

Open Access

https://doi.org/10.1038/s41467-020-19258-y

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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.

Highlights

Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases
We designed RPA primers to both the SARS-CoV-2 N gene and S gene (Supplementary Fig. 1a and Supplementary Table 1) and quantified the amplification of a reverse transcriptionrecombinase polymerase amplification (RT-RPA) assay with ProtoScript II reverse transcriptase by quantitative polymerase chain reaction (qPCR) (Supplementary Fig. 1b)
Some studies have used longer reaction times to partially counteract the poor yield of RT-RPA19, but we set out to determine whether alternative approaches were possible

Summary

Introduction

Inexpensive, robust diagnostics are essential to control the spread of infectious diseases. We report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. Efforts to increase testing capacity have included testing from saliva[4], using nonstandard storage media or dry swabs[5], and eliminating the normal RNA purification step from the standard RT-qPCR tests[6,7] Strategies such as pooling samples followed by detection using traditional or high throughput sequencing approaches have been proposed as a way to allow significantly more testing at a highly reduced cost[8,9]. Most of the approaches developed so far still require an extraction step and/or two amplification steps to achieve high specificity, or have low sensitivities that give poor concordance with the gold standard RT-qPCR test[11]. We present a method to screen for efficient RPA primers and show that proper RT enzyme selection with the addition of RNase H to a standard RPA reaction enhances detection of viral

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