Detection of SARS-CoV-2 using non-commercial RT-LAMP reagents and raw samples

Alekseenko, Alisa; Barrett, Donal; Pareja-Sanchez, Yerma; Howard, Rebecca J.; Strandback, Emilia Sofia; Ampah-Korsah, Henry; Rovšnik, Urška; Zuniga-Veliz, Silvia; Klenov, Alexander; Malloo, Jayshna; Ye, Sheng‐Long; Liu, Xiyang; Reinius, Björn; Elsässer, Simon J.; Nyman, T.; Sandh, Gustaf; Yin, Xiushan

doi:10.1101/2020.08.22.20179507

Cited by 8 publications

(16 citation statements)

References 30 publications

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“…More recently, a number of nucleic acid isothermal amplification techniques for viral detection have been described, including loop-mediated isothermal amplification (LAMP) [ [13] , [14] , [15] ], using a 4- or 6-primer set and incubated at 65 °C for 30-60 minutes, recombinase-polymerase amplification (RPA) [ 16 ], using multiple enzymes and incubated at 42 °C for 15-20 minutes, and many others [ [17] , [18] , [19] ]. Isothermal assays have been shown to detect SARS-CoV-2 with very high sensitivity and specificity, and underlie several tests recently authorized for emergency use, including the ID NOW from Abbott, and CRISPR-based detection by SHERLOCK [ 20 ] (currently not approved for sale outside the United States).…”

Section: Introductionmentioning

confidence: 99%

Extraction-free RT-LAMP to detect SARS-CoV-2 is less sensitive but highly specific compared to standard RT-PCR in 101 samples

Schellenberg¹,

Ormond²,

Keynan

2021

Journal of Clinical Virology

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The current scale of public and private testing cannot be expected to meet the emerging need for higher levels of community-level and repeated screening of asymptomatic Canadians for SARS-CoV-2. Rapid point-of-care techniques are increasingly being offered to fill the gap in screening levels required to identify undiagnosed individuals with high viral loads. However, rapid, point-of-care tests often have lower sensitivity in practice. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 has proven sensitive and specific and provides visual results in minutes. Using a commercially available kit for RT-LAMP and primer set targetting nucleocapsid (N), we tested a blinded set of 101 archived nasopharyngeal (NP) swab samples with known RT-PCR results. RT-LAMP reactions were incubated at 65 °C for 30 minutes, using heat-inactivated nasopharyngeal swab sample in viral transport medium, diluted tenfold in water, as input. RT-LAMP agreed with all RT-PCR defined negatives (N = 51), and all positives with cycle threshold (Ct) less than 20 (N = 24), 65% of positives with Ct between 20-30 (N = 17), and no positives with Ct greater than 30 (N = 9). RT-LAMP requires fewer and different core components, so may not compete directly with the mainline testing workflow, preserving precious central laboratory resources for those with the greatest need. Careful messaging must be provided when using less-sensitive tests, so that people are not falsely reassured by negative results, but this caveat must be weighed against the clear benefits of reliably identifying those with high levels of virus in prioritized samples at the point of care.

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Section: Introductionmentioning

confidence: 99%

Extraction-free RT-LAMP to detect SARS-CoV-2 is less sensitive but highly specific compared to standard RT-PCR in 101 samples

Schellenberg¹,

Ormond²,

Keynan

2021

Journal of Clinical Virology

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“…The limit of detection of ∼26 Ct (approximately 1×10 4 -1×10 5 copies/mL 45 ) and inexpensive analysis are a suitable combination for frequent screening to allow detection of the spike in viral load early upon infection and minimize the risk of transmission 16 . In the context of the current SARS-CoV-2 pandemic, the full centrifugal platform is adaptable to any smartphone, costs less than 200 USD and is compatible with cost-effective and scalable LAMP reagents under investigation 46 . These features can potentially pave the way to bring routine and scalable diagnostics to RLS, as well as expanding the current diagnostic capacities in high-income countries by bringing viral RNA detection directly to the field.…”

Section: Discussionmentioning

confidence: 99%

“…obtained nasopharyngeal samples from Karolinska University Hospital, Huddinge (Stockholm) collected between May 20 th and June 1 st 2020. Samples were collected either using Sigma-Transwab or Sigma-Virocult kits (MWE, UK) as described in detail elsewhere46 . We used pseudo-anonymized surplus material previously collected for clinical diagnostics of SARS-CoV-2.…”

mentioning

confidence: 99%

Point-of-care detection of SARS-CoV-2 in nasopharyngeal swab samples using an integrated smartphone-based centrifugal microfluidic platform

Soares

Pinto

et al. 2020

Preprint

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With its origin estimated around December 2019 in Wuhan, China, the ongoing 2020 SARS-CoV-2 pandemic is a major global health challenge, resulting in more than 45 million infections and 1.2 million deaths. The demand for scalable, rapid and sensitive viral diagnostics is thus particularly pressing at present to help contain the rapid spread of infection and prevent overwhelming the capacity of health systems. While high-income countries have managed to rapidly expand diagnostic capacities, such is not the case in resource-limited settings of low- to medium-income countries. Aiming at developing cost-effective viral load detection systems for point-of-care COVID-19 diagnostics in resource-limited and resource-rich settings alike, we report the development of an integrated modular centrifugal microfluidic platform to perform loop-mediated isothermal amplification (LAMP) of viral RNA directly from heat-inactivated nasopharyngeal swab samples. The discs were pre-packed with dried n-benzyl-n-methylethanolamine modified agarose beads used as a versatile post-nucleic acid amplification signal enhancement strategy, allowing fluorescence detection via a smartphone camera and simple optics. The platform provided sample-to-answer analysis within 1 hour from sample collection and a detection limit between 100 and 1000 RNA copies in 10 μL reaction volume. Furthermore, direct detection of non-extracted SARS-CoV-2 RNA in nasopharyngeal swab samples from patients with Ct values below 26 (n=25 plus 6 PCR negative samples) was achieved with ~94% sensitivity and 100% specificity, thus being fit-for-purpose to diagnose patients with a high risk of viral transmission. These results show significant promise towards bringing routine point-of-care COVID-19 diagnostics closer to resource-limited settings.

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“…The copyright holder for this preprint this version posted May 12, 2021. ; https://doi.org/10.1101/2021.05.08.21256891 doi: medRxiv preprint RT-PCR and RT-LAMP protocols based on homebrewed enzymes. 15,[20][21][22][23][24][25][26][27][28] Here, we describe the in-house production of RT-LAMP reactions for SARS-CoV-2 from homebrewed Moloney murine leukemia virus (M-MLV) reverse transcriptase and Bst Large Fragment (BstLF) polymerase. We compare their performance to commercial reactions and tested their ability to detect SARS-CoV-2 from RNA extracted from clinical nasopharyngeal samples.…”

Section: Introductionmentioning

confidence: 99%

Homebrew reagents for low cost RT-LAMP

Matute

Núñez

Rivera

et al. 2021

Preprint

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RT-LAMP (reverse transcription - Loop-mediated isothermal amplification) has gained popularity for the detection of SARS-CoV-2. The high specificity, sensitivity, simple protocols and potential to deliver results without the use of expensive equipment has made it an attractive alternative to RT-PCR. However, the high cost per reaction, the centralized manufacturing of required reagents and their distribution under cold chain shipping limits RT-LAMP applicability in low-income settings. The preparation of assays using homebrew enzymes and buffers has emerged worldwide as a response to these limitations and potential shortages. Here, we describe the production of Moloney murine leukemia virus (M-MLV) Reverse Transcriptase and BstLF DNA polymerase for the local implementation of RT-LAMP reactions at low cost. These reagents compared favorably to commercial kits and optimum concentrations were defined in order to reduce time to threshold, increase ON/OFF range and minimize enzyme quantities per reaction. As a validation, we tested the performance of these reagents in the detection of SARS-CoV-2 from RNA extracted from clinical nasopharyngeal samples, obtaining high agreement between RT-LAMP and RT-PCR clinical results. The in-house preparation of these reactions results in an order of magnitude reduction in costs, and thus we provide protocols and DNA to enable the replication of these tests at other locations. These results contribute to the global effort of developing open and low cost diagnostics that enable technological autonomy and distributed capacities in viral surveillance.

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Detection of SARS-CoV-2 using non-commercial RT-LAMP reagents and raw samples

Cited by 8 publications

References 30 publications

Extraction-free RT-LAMP to detect SARS-CoV-2 is less sensitive but highly specific compared to standard RT-PCR in 101 samples

Extraction-free RT-LAMP to detect SARS-CoV-2 is less sensitive but highly specific compared to standard RT-PCR in 101 samples

Point-of-care detection of SARS-CoV-2 in nasopharyngeal swab samples using an integrated smartphone-based centrifugal microfluidic platform

Homebrew reagents for low cost RT-LAMP

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