Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for point-of-care detection of SARS-CoV-2: a clinical study to evaluate agreement with RT-qPCR

Abstract

Background The global COVID-19 pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for rapid, accurate, and accessible diagnostics to enable timely treatment and outbreak control. However, current diagnostic tests based on RT-qPCR are insufficient to meet the global testing demand because of their high cost and complexity and supply chain shortages. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a promising alternative to RT-qPCR because of its sensitivity, speed, and robustness to sample inhibitors. Here, we describe the development and optimisation of a sample-to-answer workflow, including a simple lysis and inactivation protocol that provides results in <1h, using inexpensive and readily available reagents. Further, we assess the sensitivity and specificity of the developed RT-LAMP assay against a RT-qPCR. Methods We collected samples from asymptomatic healthcare workers at The University of Texas MD Anderson Cancer Center and inpatients at Lyndon B Johnson Hospital in Houston, TX, USA. Nasopharyngeal swabs were collected by medical providers and were placed directly into 300 µL of an optimised lysis buffer. Samples were heat inactivated at 95°C for 5 mins before direct amplification in a RT-LAMP assay using previously published primer sets. Heating and real-time monitoring was performed using a Bio-Rad CFX96 thermocycler and an Axxin T8-ISO, a benchtop fluorimeter designed for point-of-care settings. We compared results from the RT-LAMP test with standardof-care RT-qPCR results on paired nasopharyngeal swabs collected into Universal Viral Transport Media. Findings The developed RT-LAMP assay demonstrated a limit of detection of 4-5 virions/µL. The test requires a swab, two tubes, prepared lysis buffer, a heat block, pipettes, RT-LAMP reagents, and the real-time fluorimeter. Samples were collected between April 14, 2020 and Aug 12, 2020, and results from 74 enrolled participants were analysed in the optimised workflow. Thirty nine participants tested positive for SARS-CoV-2 and 35 participants tested negative in the hospital-administered RT-qPCR test. All 74 nasopharyngeal swab eluates were tested with our assay on the Bio-Rad CFX96; 72 nasopharyngeal swab eluates were also tested on the Axxin T8-ISO. The developed assay showed sensitivity of 92•31% and 91•89% when tested on the CFX96 and T8, respectively, and specificity of 91•43% and 97•89%, respectively. Interpretation RT-LAMP could be used for SARS-CoV-2 testing and overcomes the challenges of adapting an assay to a point-of-care instrument. Further, the reduced instrumentation cost and complexity, along with the simple workflow, highlight the potential for implementation of RT-LAMP for SARS-CoV-2 testing in resource-limited settings.

Loop-Mediated Isothermal Amplification Detection of SARS-CoV-2 and Myriad Other Applications

“…33 to 34 as the threshold for contagiousness, the totality of the data suggest that sensitivities in the 10 to 100 copies/lL range, as observed in LAMP, are well within the range required to identify clinically (and epidemiologically) relevant patients. In several recent studies (Fowler et al 2020, Howson et al 2020, Kellner et al 2020, de Oliveira Coelho 2021, Egerer et al 2021, Fowler et al 2021, Natoli 2021, Nawattanapaiboon et al 2021, Schellenberg et al 2021, RT-LAMP has generally demonstrated comparable diagnostic performance to RT-qPCR, particularly for samples with the lower, and arguably more clinically relevant, C t values in RT-PCR, and has also been shown to be close to RNA sequencing (RNA-seq) metrics (Butler et al 2021).…”

“…It has the ability to mix viscous liquids if a magnetic ball bearing is included in each tube (this feature was designed for the high-viscosity solutions used for RPA). Natoli et al (2021) describe methods used to adapt the T8 for use with LAMP at 658C (vs. RPA at 418C), particularly the need to add mineral or paraffin oil to limit evaporation because the T8 does not have a heated lid.…”

Comparative evaluation of loop‐mediated isothermal amplification and PCR for detection of Esox lucius housekeeping genes for use in on‐site environmental monitoring

“…The conventional PCR method has been extensively used in the investigation of aquatic organisms, in eDNA determination and in field studies when samples are collected for laboratory analyses (Barnes et al ., 2014; Deiner & Altermatt, 2014; Pilliod et al ., 2014). Currently, LAMP is a more suitable method for clinical diagnosis, in laboratories and in fields with limited resources (Khodaparast et al ., 2022; Lim et al ., 2021; Natoli et al ., 2021). The idea of comparing the PCR and LAMP is to find the best technique for sensitivity, less time consumption and preferably a constant temperature for an automatic lab‐on‐a‐chip analysis out in the field.…”

Programmable Digital-Microfluidic Biochips for SARS-CoV-2 Detection

“…Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) is a variant of the LAMP method that is specifically designed for the amplification of RNA sequences. It combines the principles of reverse transcription and LAMP to enable the detection of RNA viruses and other RNA sequences [ 12 , 13 , 14 , 15 ]. In the RT-LAMP process, the RNA target sequence is first reverse-transcribed into complementary DNA (cDNA) by a reverse transcriptase enzyme.…”