SARS-CoV-2 detection by a clinical diagnostic RT-LAMP assay

Buck, Michael D.; Poirier, Enzo Z.; Cardoso, Ana; Frederico, Bruno; Canton, Johnathan; Barrell, Sam; Beale, Rupert; Byrne, Richard; Caidan, Simon; Crawford, Margaret; Cubitt, Laura; Gandhi, Sonia; Goldstone, Robert; Grant, Paul; Gulati, Kiran; Hindmarsh, Steve; Howell, Michael; Hubank, Michael; Instrell, Rachael; Jiang, Ming; Kassiotis, George; Lu, Wei-Ting; MacRae, James I.; Martini, Iana; Miller, Davin; Moore, David; Nastouli, Eleni; Nicod, Jérôme; Nightingale, Luke; Olsen, Jessica; Oomatia, Amin; O’Reilly, Nicola; Rideg, Anett; Song, Ok‐Ryul; Strange, Amy; Swanton, Charles; Turajlic, Samra; Wu, Mary; Sousa, Caetano Reis e

doi:10.12688/wellcomeopenres.16517.1

Cited by 10 publications

(5 citation statements)

References 18 publications

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“…Here, we describe the development of RT‐LAMP‐based diagnostics for SARS‐CoV‐2 surveillance testing suitable for sensitive surveillance and sentinel testing in a low‐incidence period of the pandemic. Since the onset of the pandemic, a large number of publications emerged describing the use and validation of RT‐LAMP assays for SARS‐CoV‐2 diagnostics, that is (Huang et al , 2020; Klein et al , 2020; Rabe & Cepko, 2020; Alekseenko et al , 2021; Buck et al , 2021; Kundrod et al , 2022; Lu et al , 2022; Schneider et al , 2022; Zhang & Tanner, 2022), suggesting a suitable sensitivity of RT‐LAMP for the detection of infections in asymptomatic and symptomatic subjects. Here, we demonstrate the practicality of RT‐LAMP for large‐scale surveillance testing over an extended period, thus showing that RT‐LAMP is a sensitive, specific, and cost‐efficient alternative for SARS‐CoV‐2 testing.…”

Section: Discussionmentioning

confidence: 99%

Scalable RT‐LAMP‐based SARS‐CoV‐2 testing for infection surveillance with applications in pandemic preparedness

et al. 2023

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Throughout the SARS‐CoV‐2 pandemic, limited diagnostic capacities prevented sentinel testing, demonstrating the need for novel testing infrastructures. Here, we describe the setup of a cost‐effective platform that can be employed in a high‐throughput manner, which allows surveillance testing as an acute pandemic control and preparedness tool, exemplified by SARS‐CoV‐2 diagnostics in an academic environment. The strategy involves self‐sampling based on gargling saline, pseudonymized sample handling, automated RNA extraction, and viral RNA detection using a semiquantitative multiplexed colorimetric reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) assay with an analytical sensitivity comparable with RT‐qPCR. We provide standard operating procedures and an integrated software solution for all workflows, including sample logistics, analysis by colorimetry or sequencing, and communication of results. We evaluated factors affecting the viral load and the stability of gargling samples as well as the diagnostic sensitivity of the RT‐LAMP assay. In parallel, we estimated the economic costs of setting up and running the test station. We performed > 35,000 tests, with an average turnover time of < 6 h from sample arrival to result announcement. Altogether, our work provides a blueprint for fast, sensitive, scalable, cost‐ and labor‐efficient RT‐LAMP diagnostics, which is independent of potentially limiting clinical diagnostics supply chains.

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Section: Discussionmentioning

confidence: 99%

Scalable RT‐LAMP‐based SARS‐CoV‐2 testing for infection surveillance with applications in pandemic preparedness

et al. 2023

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“…However, they are prone to false positives, especially if sequence-independent readouts like a pH-sensitive dye are employed. Unfortunately, most published or commercially available RT-LAMP assays for COVID-19 rely on such readouts, with colorimetric dyes being particularly popular. − Hence, there is a need to develop more reliable assays that incorporate an extra specificity checking step to guard against false positives. A few research groups have tried to address this issue, but their target detection strategies suffer from various shortcomings, including an absence of a separate probe from the LAMP primers, nonoptimal reaction conditions, and challenging probe or riboregulator design that likely requires several iterations of testing. ,− Furthermore, the Proofman and oligonucleotide strand exchange (OSD) probe methods have not been validated with clinical samples.…”

Section: Discussionmentioning

confidence: 99%

“…Such methods typically rely on (1) the turbidity caused by precipitated magnesium pyrophosphate, (2) coffee-ring formation on colloid-crystal substrates, (3) formation of DNA-magnetic bead aggregates on filter paper, (4) melting and annealing curve analysis, (5) luciferase-catalyzed bioluminescence, (6) electrochemiluminescence, , (7) colorimetric dyes, − (8) fluorescent dyes that bind to double-stranded DNA, − or (9) agarose gel electrophoresis . Today, numerous LAMP assays for COVID-19 have been developed and commercialized based on some of these sequence-independent methods. − However, isothermal amplification often generates nonspecific products even without a template present. In particular, the large number of long primers used in LAMP results in a heightened risk of primer dimers forming.…”

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confidence: 99%

A Sensitive and Specific Fluorescent RT-LAMP Assay for SARS-CoV-2 Detection in Clinical Samples

et al. 2022

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The raging COVID-19 pandemic has created an unprecedented demand for frequent and widespread testing to limit viral transmission. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has emerged as a promising diagnostic platform for rapid detection of SARS-CoV-2, in part because it can be performed with simple instrumentation. However, isothermal amplification methods frequently yield spurious amplicons even in the absence of a template. Consequently, RT-LAMP assays can produce false positive results when they are based on generic intercalating dyes or pH-sensitive indicators. Here, we report the development of a sensitive RT-LAMP assay that leverages on a novel sequence-specific probe to guard against spurious amplicons. We show that our optimized fluorescent assay, termed LANTERN, takes only 30 min to complete and can be applied directly on swab or saliva samples. Furthermore, utilizing clinical RNA samples from 52 patients with COVID-19 infection and 21 healthy individuals, we demonstrate that our diagnostic test exhibits a specificity and positive predictive value of 95% with a sensitivity of 8 copies per reaction. Hence, our new probe-based RT-LAMP assay can serve as an inexpensive method for point-of-need diagnosis of COVID-19 and other infectious diseases.

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“…Direct detection negates the requirement for RNA extraction 23,24 , for which there has previously been competition for reagents and often requires expensive extraction equipment including liquid handling automation. This extraction-free method decreases turnaround time from sample collection to result.…”

Section: Discussionmentioning

confidence: 99%

RT-LAMP has high accuracy for detecting SARS-CoV-2 in saliva and naso/oropharyngeal swabs from asymptomatic and symptomatic individuals

Sp¹,

Burns²,

Armson³

et al. 2021

Preprint

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Previous studies have described RT-LAMP methodology for the rapid detection of SARS-CoV-2 in nasopharyngeal/oropharyngeal swab and saliva samples. Here we describe the validation of an improved simple sample preparation method for Direct SARS-CoV-2 RT-LAMP, removing the need for RNA extraction, using 559 swabs and 86,760 saliva samples from asymptomatic and symptomatic individuals across multiple healthcare settings. Using this improved method we report a diagnostic sensitivity (DSe) of 70.35% (95% CI 63.48-76.60%) on swabs and 84.62% (79.50-88.88%) on saliva, with diagnostic specificity (DSp) 100% (98.98-100.00%) on swabs and 100% (99.72-100.00%) on saliva when compared to RT-qPCR. Analysing samples with RT-qPCR ORF1ab CT values of <25 and <33 (high and medium-high viral loads, respectively), we found DSe of 100% (96.34-100%) and 77.78% (70.99-83.62%) for swabs, and 99.01% (94.61-99.97%) and 87.32% (80.71-92.31%) for saliva. We also describe RNA RT-LAMP (on extracted RNA) performed on 12,619 swabs and 12,521 saliva samples to provide updated performance data with DSe and DSp of 95.98% (92.74-98.06%) and 99.99% (99.95-100%) for swabs, and 80.65% (73.54-86.54%) and 99.99% (99.95-100%) for saliva, respectively. We also report on daily samples collected from one individual from symptom onset where both Direct and RNA RT-LAMP detected SARS-CoV-2 in saliva collected on all six days where symptoms were recorded, with RNA RT-LAMP detecting SARS-CoV-2 for an additional further day. The findings from these studies demonstrate that RT-LAMP testing of swabs and saliva is potentially applicable to a variety of use-cases, including frequent, interval-based testing of saliva from asymptomatic individuals via Direct RT-LAMP that may be missed using symptomatic testing alone.

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SARS-CoV-2 detection by a clinical diagnostic RT-LAMP assay

Cited by 10 publications

References 18 publications

Scalable RT‐LAMP‐based SARS‐CoV‐2 testing for infection surveillance with applications in pandemic preparedness

Scalable RT‐LAMP‐based SARS‐CoV‐2 testing for infection surveillance with applications in pandemic preparedness

A Sensitive and Specific Fluorescent RT-LAMP Assay for SARS-CoV-2 Detection in Clinical Samples

RT-LAMP has high accuracy for detecting SARS-CoV-2 in saliva and naso/oropharyngeal swabs from asymptomatic and symptomatic individuals

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