The ability to detect an infectious agent in a widespread epidemic is crucial to the success of quarantine efforts in addition to sensitive and accurate screening of potential cases of infection from patients in a clinical setting. Enabling testing outside of sophisticated laboratories broadens the scope of control and surveillance efforts, but also requires robust and simple methods that can be used without expensive instrumentation. Here we report a method to identify SARS-CoV-2 (COVID-19) virus RNA from purified RNA or cell lysis using loop-mediated isothermal amplification (LAMP) using a visual, colorimetric detection. This test was additionally verified using RNA samples purified from respiratory swabs collected from COVID-19 patients in Wuhan, China with equivalent performance to a commercial RT-qPCR test while requiring only heating and visual inspection. This simple and sensitive method provides an opportunity to facilitate virus detection in the field without a requirement for complex diagnostic infrastructure.

Loop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 ... METHOD SUMMARY This study describes enhancing loop-mediated isothermal amplification through the addition of guanidine chloride and the use of multiple primer sets for different gene targets combin...

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused thousands of deaths worldwide, including >18,000 in New York City (NYC) alone. The sudden emergence of this pandemic has highlighted a pressing clinical need for rapid, scalable diagnostics that can detect infection, interrogate strain evolution, and identify novel patient biomarkers. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs, plus a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, bacterial, and viral profiling. We applied both technologies across 857 SARS-CoV-2 clinical specimens and 86 NYC subway samples, providing a broad molecular portrait of the COVID-19 NYC outbreak. Our results define new features of SARS-CoV-2 evolution, nominate a novel, NYC-enriched viral subclade, reveal specific host responses in interferon, ACE, hematological, and olfaction pathways, and examine risks associated with use of ACE inhibitors and angiotensin receptor blockers. Together, these findings have immediate applications to SARS-CoV-2 diagnostics, public health, and new therapeutic targets.

The ongoing pandemic has demonstrated the utility of widespread surveillance and diagnostic detection of the novel SARS-CoV-2. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP)... METHOD SUMMARY This study describes enhancing loop-mediated isothermal amplification through multiplexed real-time and end point detection of SARS-CoV-2 combined with influenza and control targets....

The ongoing SARS-CoV-2 pandemic has necessitated a dramatic increase in our ability to conduct molecular diagnostic tests, as accurate detection of the virus is critical in preventing its spread. However, SARS-CoV-2 variants continue to emerge, with each new variant potentially affecting widely-used nucleic acid amplification diagnostic tests. RT-LAMP has been adopted as a quick, inexpensive diagnostic alternative to RT-qPCR, but as a newer method, has not been studied as thoroughly. Here we interrogate the effect of SARS-CoV-2 sequence mutations on RT-LAMP amplification, creating 523 single point mutation “variants” covering every position of the LAMP primers in 3 SARS-CoV-2 assays and analyzing their effects with over 4,500 RT-LAMP reactions. Remarkably, we observed only minimal effects on amplification speed and no effect on detection sensitivity at positions equivalent to those that significantly impact RT-qPCR assays. We also created primer sets targeting a specific short deletion and observed that LAMP is able to amplify even with a primer containing multiple consecutive mismatched bases, albeit with reduced speed and sensitivity. This highlights RT-LAMP as a robust technique for viral RNA detection that can tolerate most mutations in the primer regions. Additionally, where variant discrimination is desired, we describe the use of molecular beacons to sensitively distinguish and identify variant RNA sequences carrying short deletions. Together these data add to the growing body of knowledge on the utility of RT-LAMP and increase its potential to further our ability to conduct molecular diagnostic tests outside of the traditional clinical laboratory environment.

Prokaryotic argonautes are a unique class of nucleic acid-guided endonucleases putatively involved in cellular defense against foreign genetic elements. While their eukaryotic homologs and Cas protein counterparts require single-stranded RNAs as guides, some prokaryotic argonautes are able to utilize short single-stranded DNAs as guides for sequence-specific endonuclease activity. Many complications currently prevent the use of prokaryotic argonautes for in vivo gene-editing applications; however, they do exhibit potential as a new class of in vitro molecular tools if certain challenges can be overcome, specifically the limitations on substrate accessibility which leads to unequal levels of activity across a broad palate of substrates and the inability to act on double-stranded DNA substrates. Here we demonstrate the use of accessory factors, including thermostable single-stranded DNA binding proteins and UvrD-like helicase, in conjunction with prokaryotic argonautes to significantly improve enzymatic activity and enable functionality with a broader range of substrates, including linear double-stranded DNA substrates. We also demonstrate the use of Thermus thermophilus argonaute with accessory factors as a programmable restriction enzyme to generate long, unique single-stranded overhangs from linear double-stranded substrates compatible with downstream ligation.