Development and evaluation of a rapid and simple diagnostic assay for COVID-19 based on loop-mediated isothermal amplification

Yoshikawa, Rokusuke; Abe, Haruka; Igasaki, Yui; Negishi, Saeki; Goto, Hiroaki; Yasuda, Jiro

doi:10.1371/journal.pntd.0008855

Cited by 35 publications

(45 citation statements)

References 25 publications

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“…After these modifications, RT-LAMP achieved 77.2% overall clinical sensitivity and 97% specificity, with 93.2% sensitivity in saliva containing at least 10 viral copies/µL (Kappa = 0.895), which is on par or more sensitive compared to most of the direct RT-LAMP approaches reported so far (7,12,(22)(23)(24)(25)(26)(27)(28)(29)(30). Moreover, the high agreement (>97.6%) observed between color interpretation and specific amplification plots in rtRT-LAMP demonstrates reliability on end-point color readout if real-time analysis is not possible.…”

Section: Discussionmentioning

confidence: 96%

A novel RT-LAMP workflow for rapid salivary diagnostics of COVID-19 and effects of age, gender and time from symptom onset

Kobayashi

Brito

Moreira

et al. 2021

Preprint

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Objectives: Rapid diagnostics is pivotal to curb SARS-CoV-2 transmission, and saliva has emerged as a practical alternative to naso/oropharyngeal (NOP) specimens. We aimed to develop a direct RT-LAMP workflow for viral detection in saliva, and to provide more information regarding its potential in COVID-19 diagnostics. Methods: Clinical and contrived specimens were used to screen/optimize formulations and sample processing protocols. Salivary viral load was determined in symptomatic patients to evaluate clinical performance (n = 90) and to characterize saliva based on age, gender and time from onset of symptoms (n = 49). Results: The devised workflow achieved 93.2% sensitivity, 97% specificity, and 0.895 Kappa for salivas containing >102 copies/μL. Further analyses in saliva showed peak viral load in the first days of symptoms and lower viral loads in females, particularly among young individuals (<38 years). NOP RT-PCR data did not yield relevant associations. Conclusions: This novel saliva RT-LAMP workflow can be applied to point-of-care testing. This work reinforces that saliva better correlates with transmission dynamics than NOP specimens, and reveals gender differences that may reflect higher transmission by males. To maximize detection, testing should be done immediately after symptom onset, especially in females.

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Section: Discussionmentioning

confidence: 96%

A novel RT-LAMP workflow for rapid salivary diagnostics of COVID-19 and effects of age, gender and time from symptom onset

Kobayashi

Brito

Moreira

et al. 2021

Preprint

View full text Add to dashboard Cite

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“…An important area of ongoing development for point-of-care nucleic acid tests is rapid RNA extraction. Standard laboratory RNA extractions are very time-consuming; however, replacing an RNA puri cation step with a simple inactivation step can compromise assay sensitivity 14,23,24 . RNA puri cations result in the concentration of viral RNA and the removal of ampli cation inhibitors, both of which increase sensitivity.…”

Section: Discussionmentioning

confidence: 99%

Saliva-Dry LAMP: A Rapid Near-Patient Detection System for SARS-CoV-2.

Toppings¹,

Mohon²,

Lee³

et al. 2021

Preprint

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The highly infectious nature of SARS-CoV-2 necessitates the use of widespread testing to control the spread of the virus. Presently, the standard molecular testing method (reverse transcriptase-polymerase chain reaction, RT-PCR) is restricted to the laboratory, time-consuming, and costly. This increases the turnaround time for getting test results. The study sought to develop a rapid, near-patient saliva-based test for COVID-19 with similar accuracy to that of standard RT-PCR tests. A lyophilized dual-target reverse transcription-loop-mediated isothermal amplification (RT-LAMP) test with fluorometric detection by the naked eye. The assay relies on dry reagents that are room temperature stable. A device containing a centrifuge, heat block, and blue LED light system was manufactured to reduce the cost of performing the assay. This test has a limit of detection of 1 copy/µL and achieved positive percent agreement of 100% [95% CI 88.43% to 100.0%] and negative percent agreement of 96.7% [95% CI 82.78% to 99.92%] on saliva. Saliva-Dry LAMP can be completed in 105 minutes. Precision, cross-reactivity, and interfering substances analysis met international regulatory standards. The combination of ease of sample collection, dry reagents, visual detection, low capital equipment cost, and excellent analytical sensitivity make Saliva-Dry LAMP particularly useful for resource-limited settings.

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“…This low-cost and disposable platform incorporates lyophilized RT-LAMP reagents and produces visible results in just one hour; however, an external heating device is required for amplification [133]. Both Mautner et al and Yoshikawa et al used commercial portable heating and detection platforms for their fluorescence RT-LAMP assays which provide results in just 30 minutes with good sensitivity [134,135]. Ganguli et al developed a fully integrated smartphone platform capable of RT-LAMP amplification and real-time fluorescence monitoring via a batteryoperated heater and the smartphone camera, respectively [136]*.…”

Section: Lamp For Covid-19 Detectionmentioning

confidence: 99%

LAMP Diagnostics at the Point-of-Care: Emerging Trends and Perspectives for the Developer Community

Moehling

Choi

Dugan

et al. 2021

Expert Review of Molecular Diagnostics

160

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Introduction: Over the past decade, loop-mediated isothermal amplification (LAMP) technology has played an important role in molecular diagnostics. Amongst numerous nucleic acid amplification assays, LAMP stands out in terms of sample-to-answer time, sensitivity, specificity, cost, robustness, and accessibility, making it ideal for field-deployable diagnostics in resource-limited regions. Areas covered: In this review, we outline the front-end LAMP design practices for point-of-care (POC) applications, including sample handling and various signal readout methodologies. Next, we explore existing LAMP technologies that have been validated with clinical samples in the field. We summarize recent work that utilizes reverse transcription (RT) LAMP to rapidly detect SARS-CoV-2 as an alternative to standard PCR protocols. Finally, we describe challenges in translating LAMP from the benchtop to the field and opportunities for future LAMP assay development and performance reporting. Expert opinion: Despite the popularity of LAMP in the academic research community and a recent surge in interest in LAMP due to the COVID-19 pandemic, there are numerous areas for improvement in the fundamental understanding of LAMP, which are needed to elevate the field of LAMP assay development and characterization.

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Development and evaluation of a rapid and simple diagnostic assay for COVID-19 based on loop-mediated isothermal amplification

Cited by 35 publications

References 25 publications

A novel RT-LAMP workflow for rapid salivary diagnostics of COVID-19 and effects of age, gender and time from symptom onset

A novel RT-LAMP workflow for rapid salivary diagnostics of COVID-19 and effects of age, gender and time from symptom onset

Saliva-Dry LAMP: A Rapid Near-Patient Detection System for SARS-CoV-2.

LAMP Diagnostics at the Point-of-Care: Emerging Trends and Perspectives for the Developer Community

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