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

Abstract

Background LamPORE is a novel diagnostic platform for the detection of SARS-CoV-2 RNA combining loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyse thousands of samples per day on a single instrument. Methods We evaluated the performance of LamPORE against RT-PCR using RNA extracted from spiked respiratory samples and stored nose and throat swabs collected at two UK hospitals. Findings The limit of detection of LamPORE was ten 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 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%]). Interpretation 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.

Third-Generation Sequencing: The Spearhead towards the Radical Transformation of Modern Genomics

“…Recently, nanopore sequencing was imported to clinical diagnostics for the rapid variant detection of SARS-CoV-2 [113]. Specifically, LamPORE constitutes an in vitro diagnostic assay for accurate and scalable detection of the SARS-CoV-2 virus [114,115]. This newly introduced test has multiplexing options for simultaneous sequencing of more than 500 RNA samples with great sensitivity and specificity (>99%).…”

Molecular diagnostics of SARS-CoV-2: Findings of an international survey

“…Molecular genetic detection using nucleic acid amplification tests (NAATs), such as reverse transcriptase polymerase chain reaction (RT-PCR), are the most commonly used means of detecting infection [6] , [7] , [8] , [9] , [10] , [11] , [12] , [13] and are also considered the standard approach for identifying an infected patient. Since the first description of the virus sequence and the publication of PCR assays for early ‘in house’ adoption [14] , numerous variations of commercial and laboratory-developed-tests and -procedures (LDT/LDP) have been established [15] .…”

A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva

“…An alternative approach, ApharSeq, addresses this bottleneck by annealing barcoded RT primers to viral RNA and pooling samples prior to amplification, but the need for specialized oligo-dT magnetic beads might constitute a separate adoption barrier for this method ( Chappleboim et al, 2021 ). Finally, methods have been designed to take advantage of the extreme sensitivity and isothermal conditions of loop-mediated isothermal amplification (LAMP) ( Peto et al, 2021 ; Dao Thi et al, 2020 ; Ludwig et al, 2021 ), but they require additional manipulation to introduce barcodes ( Peto et al, 2021 ; Dao Thi et al, 2020 ) or do not allow for convenient multiplexing ( Ludwig et al, 2021 ).…”