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High-Surety Isothermal Amplification and Detection of SARS-CoV-2

Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. Howe...

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Autores principales: Bhadra, Sanchita, Riedel, Timothy E., Lakhotia, Simren, Tran, Nicholas D., Ellington, Andrew D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8265673/
https://www.ncbi.nlm.nih.gov/pubmed/34011690
http://dx.doi.org/10.1128/mSphere.00911-20
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author Bhadra, Sanchita
Riedel, Timothy E.
Lakhotia, Simren
Tran, Nicholas D.
Ellington, Andrew D.
author_facet Bhadra, Sanchita
Riedel, Timothy E.
Lakhotia, Simren
Tran, Nicholas D.
Ellington, Andrew D.
author_sort Bhadra, Sanchita
collection PubMed
description Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can often generate spurious amplicons, especially in the absence of target sequences, resulting in false-positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets and can accumulate mutations that will lead to inefficient primer binding and thus false-negative results. Multiplex assays targeting different regions of the analyte and logical signal readout using sequence-specific probes can help to reduce both false negatives and false positives. Here, we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on a non-sequence-specific readout into individual and multiplex one-pot assays that can be visually read using sequence-specific oligonucleotide strand exchange (OSD) probes. We describe both fluorescence-based and Boolean logic-gated colorimetric lateral flow readout methods and demonstrate detection of SARS-CoV-2 virions in crude human saliva. IMPORTANCE One of the key approaches to treatment and control of infectious diseases, such as COVID-19, is accurate and rapid diagnostics that is widely deployable in a timely and scalable manner. To achieve this, it is essential to go beyond the traditional gold standard of quantitative PCR (qPCR) that is often faced with difficulties in scaling due to the complexity of infrastructure and human resource requirements. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), have been long pursued as ideal, low-tech alternatives for rapid, portable testing. However, isothermal approaches often suffer from false signals due to employment of nonspecific readout methods. We describe general principles for rapidly converting nonspecifically read LAMP assays into assays that are read in a sequence-specific manner by using oligonucleotide strand displacement (OSD) probes. We also demonstrate that inclusion of OSD probes in LAMP assays maintains the simplicity of one-pot assays and a visual yes/no readout by using fluorescence or colorimetric lateral-flow dipsticks while providing accurate sequence-specific readout and the ability to logically query multiplex amplicons for redundancy or copresence. These principles not only yielded high-surety isothermal assays for SARS-CoV-2 but might also aid in the design of more sophisticated molecular assays for other analytes.
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spelling pubmed-82656732021-07-23 High-Surety Isothermal Amplification and Detection of SARS-CoV-2 Bhadra, Sanchita Riedel, Timothy E. Lakhotia, Simren Tran, Nicholas D. Ellington, Andrew D. mSphere Research Article Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can often generate spurious amplicons, especially in the absence of target sequences, resulting in false-positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets and can accumulate mutations that will lead to inefficient primer binding and thus false-negative results. Multiplex assays targeting different regions of the analyte and logical signal readout using sequence-specific probes can help to reduce both false negatives and false positives. Here, we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on a non-sequence-specific readout into individual and multiplex one-pot assays that can be visually read using sequence-specific oligonucleotide strand exchange (OSD) probes. We describe both fluorescence-based and Boolean logic-gated colorimetric lateral flow readout methods and demonstrate detection of SARS-CoV-2 virions in crude human saliva. IMPORTANCE One of the key approaches to treatment and control of infectious diseases, such as COVID-19, is accurate and rapid diagnostics that is widely deployable in a timely and scalable manner. To achieve this, it is essential to go beyond the traditional gold standard of quantitative PCR (qPCR) that is often faced with difficulties in scaling due to the complexity of infrastructure and human resource requirements. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), have been long pursued as ideal, low-tech alternatives for rapid, portable testing. However, isothermal approaches often suffer from false signals due to employment of nonspecific readout methods. We describe general principles for rapidly converting nonspecifically read LAMP assays into assays that are read in a sequence-specific manner by using oligonucleotide strand displacement (OSD) probes. We also demonstrate that inclusion of OSD probes in LAMP assays maintains the simplicity of one-pot assays and a visual yes/no readout by using fluorescence or colorimetric lateral-flow dipsticks while providing accurate sequence-specific readout and the ability to logically query multiplex amplicons for redundancy or copresence. These principles not only yielded high-surety isothermal assays for SARS-CoV-2 but might also aid in the design of more sophisticated molecular assays for other analytes. American Society for Microbiology 2021-05-19 /pmc/articles/PMC8265673/ /pubmed/34011690 http://dx.doi.org/10.1128/mSphere.00911-20 Text en Copyright © 2021 Bhadra et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Bhadra, Sanchita
Riedel, Timothy E.
Lakhotia, Simren
Tran, Nicholas D.
Ellington, Andrew D.
High-Surety Isothermal Amplification and Detection of SARS-CoV-2
title High-Surety Isothermal Amplification and Detection of SARS-CoV-2
title_full High-Surety Isothermal Amplification and Detection of SARS-CoV-2
title_fullStr High-Surety Isothermal Amplification and Detection of SARS-CoV-2
title_full_unstemmed High-Surety Isothermal Amplification and Detection of SARS-CoV-2
title_short High-Surety Isothermal Amplification and Detection of SARS-CoV-2
title_sort high-surety isothermal amplification and detection of sars-cov-2
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8265673/
https://www.ncbi.nlm.nih.gov/pubmed/34011690
http://dx.doi.org/10.1128/mSphere.00911-20
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