Direct detection of SARS-CoV-2 RNA using high-contrast pH-sensitive dyes

Brown, Timothy A.; Schaefer, Kathy; Tsang, Arthur; Yi, Hyun; Grimm, Jonathan B.; Lemire, Andrew L.; Jradi, Fadi M.; Kim, Charles; McGowan, Kevin J.; Ritola, Kimberly; Armstrong, Derek T; Mostafa, Heba H.; Korff, Wyatt; Vale, Ronald D.; Lavis, Luke D.

doi:10.1101/2020.12.26.20248878

Cited by 4 publications

(3 citation statements)

References 27 publications

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“…However, modifications of RT-LAMP-based SARS-CoV-2 assays to reduce saliva sample variability, improve result ambiguity, and increase throughput have recently been reported elsewhere and may improve the implementation of RT-LAMP-based assays for POC use. [46][47][48][49][50] In addition, we relied on a manual RT-LAMP format during POC testing. Reading assays "by eye" inevitably results in a somewhat-subjective determination of positives.…”

Section: Discussionmentioning

confidence: 99%

Initial Evaluation of a Mobile SARS-CoV-2 RT-LAMP Testing Strategy

et al. 2021

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control in the United States remains hampered, in part, by testing limitations. We evaluated a simple, outdoor, mobile, colorimetric reverse-transcription loopmediated isothermal amplification (RT-LAMP) assay workflow where self-collected saliva is tested for SARS-CoV-2 RNA. From July 16, 2020, to November 19, 2020, surveillance samples (n ¼ 4704) were collected from volunteers and tested for SARS-CoV-2 at 5 sites. Twenty-one samples tested positive for SARS-CoV-2 by RT-LAMP; 12 were confirmed positive by subsequent quantitative reverse-transcription polymerase chain reaction (qRT-PCR) testing, whereas 8 tested negative for SARS-CoV-2 RNA, and 1 could not be confirmed because the donor did not consent to further molecular testing. We estimated the false-negative rate of the RT-LAMP assay only from July 16, 2020, to September 17, 2020 by pooling residual heat-inactivated saliva that was unambiguously negative by RT-LAMP into groups of 6 or fewer and testing for SARS-CoV-2 RNA by qRT-PCR. We observed a 98.8% concordance between the RT-LAMP and qRT-PCR assays, with only 5 of 421 RT-LAMP-negative pools (2493 total samples) testing positive in the more-sensitive qRT-PCR assay. Overall, we demonstrate a rapid testing method that can be implemented outside the traditional laboratory setting by individuals with basic molecular biology skills and that can effectively identify asymptomatic individuals who would not typically meet the criteria for symptom-based testing modalities.

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Section: Discussionmentioning

confidence: 99%

Initial Evaluation of a Mobile SARS-CoV-2 RT-LAMP Testing Strategy

et al. 2021

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“…To address the color discrimination challenges associated with phenol red, Brown et al (2020) synthesized 2 pH-sensitive dyes-LAMPShade Violet (LSV) and LAMPShade Magenta (LSM)-which, respectively, offer a purple/clear and pink/clear color change to distinguish between negative and positive results. The LSV is reported to have fewer intermediate products and greater contrast, relative to phenol red, in similar minimally buffered reaction conditions (Brown et al 2020, Yu et al 2021.…”

Section: Figurementioning

confidence: 99%

Loop-Mediated Isothermal Amplification Detection of SARS-CoV-2 and Myriad Other Applications

Moore

Cahill²,

Aidelberg

et al. 2021

J Biomol Tech

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As the second year of the COVID-19 pandemic begins, it remains clear that a massive increase in the ability to test for SARS-CoV-2 infections in a myriad of settings is critical to controlling the pandemic and to preparing for future outbreaks. The current gold standard for molecular diagnostics is the polymerase chain reaction (PCR), but the extraordinary and unmet demand for testing in a variety of environments means that both complementary and supplementary testing solutions are still needed. This review highlights the role that loop-mediated isothermal amplification (LAMP) has had in filling this global testing need, providing a faster and easier means of testing, and what it can do for future applications, pathogens, and the preparation for future outbreaks. This review describes the current state of the art for research of LAMP-based SARS-CoV-2 testing, as well as its implications for other pathogens and testing. The authors represent the global LAMP (gLAMP) Consortium, an international research collective, which has regularly met to share their experiences on LAMP deployment and best practices; sections are devoted to all aspects of LAMP testing, including preanalytic sample processing, target amplification, and amplicon detection, then the hardware and software required for deployment are discussed, and finally, a summary of the current regulatory landscape is provided. Included as well are a series of first-person accounts of LAMP method development and deployment. The final discussion section provides the reader with a distillation of the most validated testing methods and their paths to implementation. This review also aims to provide practical information and insight for a range of audiences: for a research audience, to help accelerate research through sharing of best practices; for an implementation audience, to help get testing up and running quickly; and for a public health, clinical, and policy audience, to help convey the breadth of the effect that LAMP methods have to offer.

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“…For example, researchers have explored several indirect approaches for the visualization of LAMP reaction by making use of metal chelating dyes, pH-sensitive fluorophores, and turbidity measurements of the final product. 34,35 However, most organic fluorophores are not stable and suffer from poor quantum yields; thus, the scope for improvement persists. In contrast, CDs have a larger surface area, superior photophysical properties, and photostability which could be effectively utilized in monitoring PPi in LAMP reactions.…”

Section: ■ Introductionmentioning

confidence: 99%

Dual-Emissive Carbon Dots: Exploring Their Fluorescence Properties for Sensitive Turn-Off–On Recognition of Ferric and Pyrophosphate Ions and Its Application in Fluorometric Detection of the Loop-Mediated Isothermal Amplification Reaction

et al. 2023

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In this study, dual-emissive carbon dots (CDs) were prepared using p-phenylenediamine (pPDA) and phytic acid (PA) precursors via a one-pot-hydrothermal method. The photophysical, morphological, and structural characterization of CDs was carried out using absorption, fluorescence, Fourier transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (NMR), and high-resolution transmission electron microscopy (HR-TEM) analysis. The as-prepared CDs displayed dual-fluorescence peaks at 525 and 620 nm upon excitation at 450 nm. The CDs showed good photostability and exhibited solvent-dependent fluorescence properties. The solvatochromic behavior of CDs was utilized to detect water content in organic solvents. Furthermore, the dual-emissive property of CDs was utilized for the sequential detection of ferric (Fe 3+ ) and pyrophosphate ions (PPi) by a fluorescence turn-off−on mechanism. The proposed assay showed appreciable fluorescence response toward Fe 3+ and PPi with high selectivity and good tolerance for common interfering ions. The potential practical application of the CD probe was ascertained by carrying out the fluorometric detection of PPi to affirm the loop-mediated isothermal amplification (LAMP) reaction specific for Mycobacterium tuberculosis (negative and positive clinical samples).

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Direct detection of SARS-CoV-2 RNA using high-contrast pH-sensitive dyes

Cited by 4 publications

References 27 publications

Initial Evaluation of a Mobile SARS-CoV-2 RT-LAMP Testing Strategy

Initial Evaluation of a Mobile SARS-CoV-2 RT-LAMP Testing Strategy

Loop-Mediated Isothermal Amplification Detection of SARS-CoV-2 and Myriad Other Applications

Dual-Emissive Carbon Dots: Exploring Their Fluorescence Properties for Sensitive Turn-Off–On Recognition of Ferric and Pyrophosphate Ions and Its Application in Fluorometric Detection of the Loop-Mediated Isothermal Amplification Reaction

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