SARS-CoV-2 detection by fluorescence loop-mediated isothermal amplification with and without RNA extraction

Taki, Keisuke; Yokota, Isao; Fukumoto, Tatsuya; Iwasaki, Shinya; Fujisawa, Shinichi; Takahashi, Masayoshi; Negishi, Saeki; Hayasaka, Kasumi; Sato, Kaori; Oguri, Satoshi; Nishida, Mutsumi; Sato, Junichi; Konno, Satoshi; Saito, Tomoya; Teshima, Takanori

doi:10.1016/j.jiac.2020.10.029

Cited by 35 publications

(36 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Reverse transcriptase loop-mediated isothermal ampli fication (LAMP) 25 has become the second most common NAAT after RT-qPCR with several advantages over RT-qPCR, including rapid turnaround time within 30 min, ease of implementation, and potential utility at the point of care with a simple device. 11,14,[26][27][28][29][30][31] In 2020 we reported that LAMP had equivalent efficacy to RT-qPCR in detecting SAR-CoV-2 when testing saliva from asymptomatic people in our contact tracing and airport quarantine cohorts. 11 For these reasons, LAMP is currently being used at the international airport quarantine stations in Japan as the confirmatory NAAT test after indeterminate results from CLEIA.…”

Section: Discussionmentioning

confidence: 94%

A novel strategy for SARS-CoV-2 mass screening with quantitative antigen testing of saliva: a diagnostic accuracy study

Yokota¹,

Shane²,

Okada³

et al. 2021

The Lancet Microbe

Self Cite

View full text Add to dashboard Cite

Background Quantitative RT-PCR (RT-qPCR) of nasopharyngeal swab (NPS) samples for SARS-CoV-2 detection requires medical personnel and is time consuming, and thus is poorly suited to mass screening. In June, 2020, a chemiluminescent enzyme immunoassay (CLEIA; Lumipulse G SARS-CoV-2 Ag kit, Fujirebio, Tokyo, Japan) was developed that can detect SARS-CoV-2 nucleoproteins in NPS or saliva samples within 35 min. In this study, we assessed the utility of CLEIA in mass SARS-CoV-2 screening. MethodsWe did a diagnostic accuracy study to develop a mass-screening strategy for salivary detection of SARS-CoV-2 by CLEIA, enrolling hospitalised patients with clinically confirmed COVID-19, close contacts identified at community health centres, and asymptomatic international arrivals at two airports, all based in Japan. All test participants were enrolled consecutively. We assessed the diagnostic accuracy of CLEIA compared with RT-qPCR, estimated according to concordance (Kendall's coefficient of concordance, W), and sensitivity (probability of CLEIA positivity given RT-qPCR positivity) and specificity (probability of CLEIA negativity given RT-qPCR negativity) for different antigen concentration cutoffs (0•19 pg/mL, 0•67 pg/mL, and 4•00 pg/mL; with samples considered positive if the antigen concentration was equal to or more than the cutoff and negative if it was less than the cutoff). We also assessed a two-step testing strategy post hoc with CLEIA as an initial test, using separate antigen cutoff values for test negativity and positivity from the predefined cutoff values. The proportion of intermediate results requiring secondary RT-qPCR was then quantified assuming prevalence values of RT-qPCR positivity in the overall tested population of 10%, 30%, and 50%. Findings Self-collected saliva was obtained from 2056 participants between June 12 and Aug 6, 2020. Results of CLEIA and RT-qPCR were concordant in 2020 (98•2%) samples (Kendall's W=0•99). Test sensitivity was 85•4% (76 of 89 positive samples; 90% credible interval [CrI] 78•0-90•3) at the cutoff of 0•19 pg/mL; 76•4% (68 of 89; 68•2-82•8) at the cutoff of 0•67 pg/mL; and 52•8% (47 of 89; 44•1-61•3) at the cutoff of 4•0 pg/mL. Test specificity was 91•3% (1796 of 1967 negative samples; 90% CrI 90•2-92•3) at the cutoff of 0•19 pg/mL, 99•2% (1952 of 1967; 98•8-99•5) at the cutoff of 0•67 pg/mL, and 100•0% (1967 of 1967; 99•8-100•0) at the cutoff of 4•00 pg/mL. Using a two-step testing strategy with a CLEIA negativity cutoff of 0•19 pg/mL (to maximise sensitivity) and a CLEIA positivity cutoff of 4•00 pg/mL (to maximise specificity), the proportions of indeterminate results (ie, samples requiring secondary RT-qPCR) would be approximately 11% assuming a prevalence of RT-qPCR positivity of 10%, 16% assuming a prevalence of RT-qPCR positivity of 30%, and 21% assuming a prevalence of RT-qPCR positivity of 50%.Interpretation CLEIA testing of self-collected saliva is simple and provides results quickly, and is thus suitable for mass testing. To improve accuracy, we propose a two-step ...

show abstract

Section: Discussionmentioning

confidence: 94%

A novel strategy for SARS-CoV-2 mass screening with quantitative antigen testing of saliva: a diagnostic accuracy study

Yokota¹,

Shane²,

Okada³

et al. 2021

The Lancet Microbe

Self Cite

View full text Add to dashboard Cite

show abstract

“…Compared to nasopharyngeal swabs, saliva samples harbor similar levels of viral load while being easier to obtain via self-collection. Several groups have developed RT-LAMP tests to detect SARS-CoV-2 in saliva samples ( Bhadra et al, 2020 ; Flynn et al, 2020 ; Lalli et al, 2020 ; Lamb et al, 2020 ; Nagura-Ikeda et al, 2020 ; Rabe and Cepko, 2020 ; Taki et al, 2021 ; Yokota et al, 2020 ). However, due to pH variability between saliva samples, RT-LAMP often has a high rate of false positives when used with the common pH-dependent dye phenol red ( Bhadra et al, 2020 ; Hardinge and Murray, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers

Yang

Meyerson

Clark

et al. 2021

eLife

View full text Add to dashboard Cite

Here, we develop a simple molecular test for SARS-CoV-2 in saliva based on reverse transcription loop-mediated isothermal amplification (RT-LAMP). The test has two steps: 1) heat saliva with a stabilization solution, and 2) detect virus by incubating with a primer/enzyme mix. After incubation, saliva samples containing the SARS-CoV-2 genome turn bright yellow. Because this test is pH dependent, it can react falsely to some naturally acidic saliva samples. We report unique saliva stabilization protocols that rendered 295 healthy saliva samples compatible with the test, producing zero false positives. We also evaluated the test on 278 saliva samples from individuals who were infected with SARS-CoV-2 but had no symptoms at the time of saliva collection, and from 54 matched pairs of saliva and anterior nasal samples from infected individuals. The Saliva TwoStep test described herein identified infections with 94% sensitivity and >99% specificity in individuals with sub-clinical (asymptomatic or pre-symptomatic) infections.

show abstract

“…In the initial months of the pandemic, preprints were released that highlighted alternative strategies apart from RT-qPCR for SARS-CoV-2 detection [10][11][12][13][14][15][16][17] , although robust validation of these techniques using bona fide COVID-19 positive clinical specimens was sometimes lacking. We were able to validate that RT-LAMP could be a specific, reliable, and inexpensive assay, albeit less sensitive, with clinical confirmation by cross comparison with our newly established RT-qPCR pipeline.…”

Section: Discussionmentioning

confidence: 99%

“…The strand displacement and amplification procedure is carried out at a single temperature in less than 30 minutes. Diagnostic tests utilising this technique have been developed for RNA viruses and other pathogens [7][8][9] , and there are now several works focusing on the use of RT-LAMP to detect SARS-CoV-2 [10][11][12][13][14][15][16][17] . We set up a SARS-CoV-2 RT-LAMP assay using the WarmStart Colorimetric LAMP 2X Mastermix commercialised by New England Biolabs (NEB), which allows for visual assessment of DNA amplification.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

View full text Add to dashboard Cite

The ongoing pandemic of SARS-CoV-2 calls for rapid and cost-effective methods to accurately identify infected individuals. The vast majority of patient samples is assessed for viral RNA presence by RT-qPCR. Our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than 252,000 RT-qPCR results since its commencement at the beginning of April 2020. However, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. In this work, we present a clinically-validated procedure for high-throughput SARS-CoV-2 detection by RT-LAMP that is robust, reliable, repeatable, specific, and inexpensive.

show abstract

SARS-CoV-2 detection by fluorescence loop-mediated isothermal amplification with and without RNA extraction

Cited by 35 publications

References 14 publications

A novel strategy for SARS-CoV-2 mass screening with quantitative antigen testing of saliva: a diagnostic accuracy study

A novel strategy for SARS-CoV-2 mass screening with quantitative antigen testing of saliva: a diagnostic accuracy study

Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers

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

Contact Info

Product

Resources

About