Diagnostic accuracy of Loop mediated isothermal amplification coupled to Nanopore sequencing for the detection of SARS-CoV-2 infection at scale in symptomatic and asymptomatic populations

Ptasińska, Anetta; Whalley, Celina M.; Bosworth, Andrew; Poxon, Charlotte; Bryer, Claire; Machin, Nicholas; Grippon, Seden; Wise, Emma L.; Armson, Bryony; Howson, Emma L.A.; Goring, Alice; Snell, Gemma; Forster, Jade; Mattocks, Christopher; Frampton, Sarah E.; Anderson, Rebecca; Cleary, David W.; Parker, Joe; Boukas, Konstantinos; Graham, Nichola; Cellura, Doriana; Garratt, Emma; Skilton, Rachel J.; Sheldon, Hana; Collins, Alla; Ahmad, Nusreen; Friar, Simon; Williams, Tim; Godfrey, Keith M.; Deans, Zandra C.; Douglas, Angela; Hill, Sue; Kidd, Michael; Porter, Deborah; Kidd, Stephen P.; Cortes, Nicholas; Fowler, Veronica L.; Williams, Tony; Richter, Alex; Beggs, Andrew D.

doi:10.1101/2020.12.15.20247031

Cited by 5 publications

(6 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Sensitivity and specificity of RT-LAMP assays should be evaluated against a range of SARS-CoV-2 viral loads for validation and optimization. LAMP has recently been coupled with nanopore sequencing and CRISPR-based detection platforms (explained below) to boost accuracy and performance (183,184).…”

Section: Other Methods Of Viral Rna Detectionmentioning

confidence: 99%

“…Real-time results are monitored with colorimetric or fluorescent dyes (43,180) • False positives may occur due to presence of multiple pair primers (183), while false-negatives may occur with low viral RNA (175,183); indicates evaluation should be performed across a range of SARS-CoV-2 viral loads • Smartphone integration and combination with nanopore sequencing and CRISPR-based detection platforms may improve performance (183,184,313) (Continued)…”

Section: In-vitro Diagnostics: Antigen Testingmentioning

confidence: 99%

See 1 more Smart Citation

Review of Current COVID-19 Diagnostics and Opportunities for Further Development

Mardian¹,

Kosasih²,

et al. 2021

View full text Add to dashboard Cite

Diagnostic testing plays a critical role in addressing the coronavirus disease 2019 (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Rapid and accurate diagnostic tests are imperative for identifying and managing infected individuals, contact tracing, epidemiologic characterization, and public health decision making. Laboratory testing may be performed based on symptomatic presentation or for screening of asymptomatic people. Confirmation of SARS-CoV-2 infection is typically by nucleic acid amplification tests (NAAT), which requires specialized equipment and training and may be particularly challenging in resource-limited settings. NAAT may give false-negative results due to timing of sample collection relative to infection, improper sampling of respiratory specimens, inadequate preservation of samples, and technical limitations; false-positives may occur due to technical errors, particularly contamination during the manual real-time polymerase chain reaction (RT-PCR) process. Thus, clinical presentation, contact history and contemporary phyloepidemiology must be considered when interpreting results. Several sample-to-answer platforms, including high-throughput systems and Point of Care (PoC) assays, have been developed to increase testing capacity and decrease technical errors. Alternatives to RT-PCR assay, such as other RNA detection methods and antigen tests may be appropriate for certain situations, such as resource-limited settings. While sequencing is important to monitor on-going evolution of the SARS-CoV-2 genome, antibody assays are useful for epidemiologic purposes. The ever-expanding assortment of tests, with varying clinical utility, performance requirements, and limitations, merits comparative evaluation. We herein provide a comprehensive review of currently available COVID-19 diagnostics, exploring their pros and cons as well as appropriate indications. Strategies to further optimize safety, speed, and ease of SARS-CoV-2 testing without compromising accuracy are suggested. Access to scalable diagnostic tools and continued technologic advances, including machine learning and smartphone integration, will facilitate control of the current pandemic as well as preparedness for the next one.

show abstract

Section: Other Methods Of Viral Rna Detectionmentioning

confidence: 99%

Section: In-vitro Diagnostics: Antigen Testingmentioning

confidence: 99%

Review of Current COVID-19 Diagnostics and Opportunities for Further Development

Mardian¹,

Kosasih²,

et al. 2021

View full text Add to dashboard Cite

show abstract

“…As expected, methods that reported detection sensitivity close to RT-qPCR tests (200-1,000 gce/ml) mostly followed traditional barcoding and sequencing workflows, and required individual RNA extraction and PCR thermocycling steps (Fig. S1) (12)(13)(14)17) (or used an extraction-free protocol but with ~10 fold lower sensitivity (12,19)), which in practice hinders the maximum achievable sample throughput (Fig. 1A).…”

Section: Introductionmentioning

confidence: 68%

“…In principle the very high throughput readout (up to 10 10 reads per session, on an Illumina NovaSeq machine) would allow a single testing lab to process up to a million patient samples per day with pooled analysis, if they could avoid the handling of so many individual samples. Since the beginning of the COVID-19 pandemic, several methods for NGS-based multiplexed testing have been proposed and developed (12)(13)(14)(15)(16)(17)(18). As expected, methods that reported detection sensitivity close to RT-qPCR tests (200-1,000 gce/ml) mostly followed traditional barcoding and sequencing workflows, and required individual RNA extraction and PCR thermocycling steps (Fig.…”

Section: Introductionmentioning

confidence: 99%

One-Seq: A Highly Scalable Sequencing-Based Diagnostic for SARS-CoV-2 and Other Single-Stranded Viruses

Dai

Kang

et al. 2021

Preprint

View full text Add to dashboard Cite

The management of pandemics such as COVID-19 requires highly scalable and sensitive viral diagnostics, together with variant identification. Next-generation sequencing (NGS) has many attractive features for highly multiplexed testing, however current sequencing-based methods are limited in throughput by early processing steps on individual samples (e.g. RNA extraction and PCR amplification). Here we report a new method, "One-Seq", that eliminates the current bottlenecks in scalability by enabling early pooling of samples, before any extraction or amplification steps. To enable early pooling, we developed a one-pot reaction for efficient reverse transcription (RT) and upfront barcoding in extraction-free clinical samples, and a "protector" strategy in which carefully designed competing oligonucleotides prevent barcode crosstalk and preserve detection of the high dynamic range of viral load in clinical samples. This method is highly sensitive, achieving a limit of detection (LoD) down to 2.5 genome copy equivalent (gce) in contrived RT samples, 10 gce in multiplexed sequencing, and 2-5 gce with multi-primer detection, suggesting an LoD of 200-500 gce/ml for clinical testing. In clinical specimens, One-Seq showed quantitative viral detection against clinical Ct values with 6 logs of linear dynamic range and detection of SARS-CoV-2 positive samples down to ~360 gce/ml. In addition, One-Seq reports a number of hotspot viral mutations at equal scalability at no extra cost. Scaling up One-Seq would allow a throughput of 100,000-1,000,000 tests per day per single clinical lab, at an estimated amortized reagent cost of $1.5 per test and turn-around time of 7.5-15 hr.

show abstract

“…The LamPORE SARS-CoV-2 assay utilises a combination of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and Nanopore sequencing technology (Oxford Nanopore Technologies, Oxford), as described previously 16 , 17 . For the SARS-CoV-2 the target regions N2 , E1 and ORF1a genes each span approximately 180bp of the viral genome.…”

Section: Methodsmentioning

confidence: 99%

Multi-modality detection of SARS-CoV-2 in faecal donor samples for transplantation and in asymptomatic emergency surgical admissions

et al. 2021

Self Cite

View full text Add to dashboard Cite

Background: Faecal transplantation is an evidence-based treatment for Clostridioides difficile. Patients infected with SARS-CoV-2 have been shown to shed the virus in stool for up to 33 days, well beyond the average clearance time for upper respiratory tract shedding. We carried out an analytical and clinical validation of reverse-transcriptase quantitative (RT-qPCR) as well as LAMP, LamPORE and droplet digital PCR in the detection of SARS-CoV-2 RNA in stool from donated samples for faecal microbiota transplantation (FMT), spiked samples and asymptomatic inpatients in an acute surgical unit. Methods: Killed SARS-CoV-2 viral lysate and extracted RNA was spiked into donor stool & FMT and a linear dilution series from 10-1 to 10-5 and tested via RT-qPCR, LAMP, LamPORE and ddPCR against SARS-CoV-2. Patients admitted to the critical care unit with symptomatic SARS-CoV-2 and sequential asymptomatic patients from acute presentation to an acute surgical unit were also tested. Results: In a linear dilution series, detection of the lowest dilution series was found to be 8 copies per microlitre of sample. Spiked lysate samples down to 10-2 dilution were detected in FMT samples using RTQPCR, LamPORE and ddPCR and down to 10-1 with LAMP. In symptomatic patients 5/12 had detectable SARS-CoV-2 in stool via RT-qPCR and 6/12 via LamPORE, and in 1/97 asymptomatic patients via RT-qPCR. Conclusion: RT-qPCR can be detected in FMT donor samples using RT-qPCR, LamPORE and ddPCR to low levels using validated pathways. As previously demonstrated, nearly half of symptomatic and less than one percent of asymptomatic patients had detectable SARS-CoV-2 in stool.

show abstract

Diagnostic accuracy of Loop mediated isothermal amplification coupled to Nanopore sequencing for the detection of SARS-CoV-2 infection at scale in symptomatic and asymptomatic populations

Cited by 5 publications

References 17 publications

Review of Current COVID-19 Diagnostics and Opportunities for Further Development

Review of Current COVID-19 Diagnostics and Opportunities for Further Development

One-Seq: A Highly Scalable Sequencing-Based Diagnostic for SARS-CoV-2 and Other Single-Stranded Viruses

Multi-modality detection of SARS-CoV-2 in faecal donor samples for transplantation and in asymptomatic emergency surgical admissions

Contact Info

Product

Resources

About