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. 2021 May 19;59(6):e03271-20.
doi: 10.1128/JCM.03271-20. Print 2021 May 19.

Diagnosis of SARS-CoV-2 Infection with LamPORE, a High-Throughput Platform Combining Loop-Mediated Isothermal Amplification and Nanopore Sequencing

Leon Peto #  1   2 , Gillian Rodger #  2 , Daniel P Carter #  3 , Karen L Osman  3 , Mehmet Yavuz  4 , Katie Johnson  4 , Mohammad Raza  4 , Matthew D Parker  5 , Matthew D Wyles  5 , Monique Andersson  6 , Anita Justice  6 , Alison Vaughan  2 , Sarah Hoosdally  2 , Nicole Stoesser  6   2 , Philippa C Matthews  6   2 , David W Eyre  6   2   7 , Timothy E A Peto  6   2 , Miles W Carroll  3   8 , Thushan I de Silva  4   9 , Derrick W Crook  6   2   10 , Cariad M Evans #  4 , Steven T Pullan #  3
Affiliations

Diagnosis of SARS-CoV-2 Infection with LamPORE, a High-Throughput Platform Combining Loop-Mediated Isothermal Amplification and Nanopore Sequencing

Leon Peto et al. J Clin Microbiol. .

Abstract

LamPORE is a novel diagnostic platform for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA combining loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyze thousands of samples per day on a single instrument. We evaluated the performance of LamPORE against reverse transcriptase PCR (RT-PCR) using RNA extracted from spiked respiratory samples and stored nose and throat swabs collected at two UK hospitals. The limit of detection of LamPORE was 10 genome copies/μl of extracted RNA, which is above the limit achievable by RT-PCR, but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among them, LamPORE had a diagnostic sensitivity of 99.1% (226/228; 95% confidence interval [CI], 96.9% to 99.9%). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279; 98.0% to 100.0%). Overall, 1.4% (7/514; 0.5% to 2.9%) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478/494; 94.8% to 98.1%). LamPORE has a similar performance as RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients and offers a promising approach to high-throughput testing.

Keywords: LamPORE; SARS-CoV-2; diagnosis; nanopore sequencing.

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Figures

FIG 1
FIG 1
Analytical specificity of LamPORE in samples positive for a range of respiratory pathogens. Data for both LamPORE replicates are shown. The dashed line is the threshold for a positive result (≥50 reads), and the dotted line is the threshold for an inconclusive result (≥20 reads). “Other pathogens” includes parainfluenza virus (n = 10), Mycoplasma pneumoniae (n = 3), and human metapneumovirus (n = 1). Invalid samples are plotted.
FIG 2
FIG 2
Total number of reads assigned to SARS-CoV-2 targets by LamPORE versus RT-PCR E gene CT value. The dashed line is the threshold for a positive result (≥50 reads), and the dotted line is the threshold for an inconclusive result (≥20 reads). Results are for replicate 1 only. Invalid samples are not shown.
See this image and copyright information in PMC

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