Development and validation of direct RT-LAMP for SARS-CoV-2

Mohon, Abu Naser; Hundt, Jana; Marle, Guido van; Berenger, Byron M.; Griener, Thomas P.; Lisboa, Luiz F.; Church, Deirdre L.; Czub, Markus; Greninger, Alexander L.; Jerome, Keith R.; Doolan, Cody; Pillai, Dylan R.

doi:10.1101/2020.04.29.20075747

Cited by 19 publications

(17 citation statements)

References 19 publications

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“…Working with extracted purified RNA, they reach a sensitivity of 97.5% for samples with RT-qPCR Cq values until 30. The same range of sensitivity is given by another study with 97.62% positive percent agreement in RT-LAMP detecting SARS-CoV-2 [ 22 ]. However Thi et al [ 21 ] tested a similar pre-treatment condition for using direct pharyngeal swab samples and avoid RNA extraction in RT-LAMP, but utilised the less suitable colorimetric read-out, with lower sensitivity yield.…”

Section: Discussionmentioning

confidence: 52%

Rapid point-of-care detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Mautner¹,

Baillie²,

Herold³

et al. 2020

Virol J

114

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Background Fast, reliable and easy to handle methods are required to facilitate urgently needed point-of-care testing (POCT) in the current coronavirus pandemic. Life-threatening severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread all over the world, infecting more than 33,500,000 people and killing over 1 million of them as of October 2020. Infected individuals without any symptoms might still transfer the virus to others underlining the extraordinary transmissibility of this new coronavirus. In order to identify early infections effectively, treat patients on time and control disease spreading, rapid, accurate and onsite testing methods are urgently required. Results Here we report the development of a loop-mediated isothermal amplification (LAMP) based method to detect SARS-CoV-2 genes ORF8 and N directly from pharyngeal swab samples. The established reverse transcription LAMP (RT-LAMP) assay detects SARS-CoV-2 directly from pharyngeal swab samples without previous time-consuming and laborious RNA extraction. The assay is sensitive and highly specific for SARS-CoV-2 detection, showing no cross reactivity when tested on 20 other respiratory pathogens. The assay is 12 times faster and 10 times cheaper than routine reverse transcription real-time polymerase chain reaction, depending on the assay used. Conclusion The fast and easy to handle RT-LAMP assay amplifying specifically the genomic regions ORF8 and N of SARS-CoV-2 is ideally suited for POCT at e.g. railway stations, airports or hospitals. Given the current pandemic situation, rapid, cost efficient and onsite methods like the here presented RT-LAMP assay are urgently needed to contain the viral spread.

show abstract

Section: Discussionmentioning

confidence: 52%

Rapid point-of-care detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Mautner¹,

Baillie²,

Herold³

et al. 2020

Virol J

114

View full text Add to dashboard Cite

show abstract

“…Although there have been recent reports of RT-LAMP−based assays to detect SARS-CoV-2 (39,40), to the best of our knowledge, no previous study has shown detection of SARS-CoV-2 using a handheld portable instrument with an integrated disposable cartridge without RNA extraction. Other relevant technologies, such as the assay from Color Genomics, report high throughput with a low LOD (0.75 copies per μL) but require a bead-based RNA extraction step (9).…”

Section: Discussionmentioning

confidence: 96%

Rapid isothermal amplification and portable detection system for SARS-CoV-2

Ganguli

Mostafa

Berger

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

353

287

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The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

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“…Miniaturizing LAMP and CRISPR reactions results in a slight loss of sensitivity and therefore may not be suitable for making diagnostic decisions where qPCR capacity is available; however, their isothermal incubation allows for thousands of samples to be tested simultaneously (Comparison of Methodologies in Supplementary Table 2). LAMP is a particularly attractive technique, because it has also been shown to be sensitive with heat-inactivated samples, removing the bottleneck of RNA extraction 27 . Furthermore, these solutions can be deployed for community testing in low-resource settings or at the point-of-care without expensive equipment requirements.…”

Section: Discussionmentioning

confidence: 99%

A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics

et al. 2020

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The SARS-CoV-2 pandemic has shown how a rapid rise in demand for patient and community sample testing can quickly overwhelm testing capability globally. With most diagnostic infrastructure dependent on specialized instruments, their exclusive reagent supplies quickly become bottlenecks, creating an urgent need for approaches to boost testing capacity. We address this challenge by refocusing the London Biofoundry onto the development of alternative testing pipelines. Here, we present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly deployed and scaled. Using an in-house-generated, open-source, MS2-virus-like particle (VLP) SARS-CoV-2 standard, we validate RNA extraction and RT-qPCR workflows as well as two detection assays based on CRISPR-Cas13a and RT-loop-mediated isothermal amplification (RT-LAMP). In collaboration with an NHS diagnostic testing lab, we report the performance of the overall workflow and detection of SARS-CoV-2 in patient samples using RT-qPCR, CRISPR-Cas13a, and RT-LAMP. The validated RNA extraction and RT-qPCR platform has been installed in NHS diagnostic labs, increasing testing capacity by 1000 samples per day.

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Development and validation of direct RT-LAMP for SARS-CoV-2

Cited by 19 publications

References 19 publications

Rapid point-of-care detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Rapid point-of-care detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Rapid isothermal amplification and portable detection system for SARS-CoV-2

A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics

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