Diagnosis of SARS-CoV-2 infection with LamPORE, a high-throughput platform combining loop-mediated isothermal amplification and nanopore sequencing

Abstract

LamPORE is a novel diagnostic platform for the detection of SARS-CoV-2 RNA that combines loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyse thousands of samples per day on a single instrument. We evaluated the performance of LamPORE against RT-PCR using RNA extracted from spiked respiratory samples and from stored nose and throat swabs collected at two UK hospitals. The limit of detection of LamPORE was 7-10 genome copies/microlitre of extracted RNA. This 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 these LamPORE had a diagnostic sensitivity of 99.1% (226/228 [95% CI 96.9-99.9%]). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279 [98.0-100.0%]). Overall, 1.4% (7/514 [0.5-2.9]) 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 [94.8-98.1]). This indicates that LamPORE has a similar performance to RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients, and offers a promising approach to high-throughput testing.

Increasing SARS-CoV-2 RT-qPCR testing capacity by sample pooling

“…Therefore, our data support that RT-qPCR testing on pooled swab samples before RNA extraction is accurate, sensitive, and feasible as an option to efficiently increase SARS-CoV-2 testing capacity and for preparedness to test large amounts of people to control virus community transmission and sudden uncontrolled outbreaks. Pooling with compressive sampling designs or hypercube slicing algorithms that involve repetitive tests (Shental et al 2020;Mutesa et al 2020), pooling of alternative less-invasive samples from the oral cavity (Watkins et al 2020), and the use of alternative testing approximations (Peto et al 2020) may help to further lessen the impact of the dilution factor on pooling and continue increasing health capacity building.…”

LamPORE: rapid, accurate and highly scalable molecular screening for SARS-CoV-2 infection, based on nanopore sequencing

“…The resulting combination, LamPORE, is rapid, sensitive and highly scalable and here we demonstrate LamPORE’s efficacy for detecting the presence or absence of SARS-CoV-2 RNA in clinical samples. Studies using much larger sample sets have been recently conducted to establish diagnostic performance claims (20).…”

Characterising Foot-and-Mouth Disease Virus in Clinical Samples Using Nanopore Sequencing

“…The use of the MinION sequencer could be investigated in conjunction with a range of protocols and equipment that have enabled the mobilisation of laboratory procedures and shorten turnaround times, for example a portable PCR platform and the rapid sequencing kit or lyophilised field sequencing kits that are now available from ONT (41). Alternatively the use of lamPORE, which combines RT-LAMP with the rapid sequencing kit and has recently been described for SARS-CoV-2 detection, could be investigated for its suitably for FMD detection and characterisation (42,43).…”