Development and Clinical Validation of RT-LAMP-Based Lateral-Flow Devices and Electrochemical Sensor for Detecting Multigene Targets in SARS-CoV-2

Abstract

Consistently emerging variants and the life-threatening consequences of SARS-CoV-2 have prompted worldwide concern about human health, necessitating rapid and accurate point-of-care diagnostics to limit the spread of COVID-19. Still, However, the availability of such diagnostics for COVID-19 remains a major rate-limiting factor in containing the outbreaks. Apart from the conventional reverse transcription polymerase chain reaction, loop-mediated isothermal amplification-based (LAMP) assays have emerged as rapid and efficient systems to detect COVID-19. The present study aims to develop RT-LAMP-based assay system for detecting multiple targets in N, ORF1ab, E, and S genes of the SARS-CoV-2 genome, where the end-products were quantified using spectrophotometry, paper-based lateral-flow devices, and electrochemical sensors. The spectrophotometric method shows a LOD of 10 agµL−1 for N, ORF1ab, E genes and 100 agµL−1 for S gene in SARS-CoV-2. The developed lateral-flow devices showed an LOD of 10 agµL−1 for all four gene targets in SARS-CoV-2. An electrochemical sensor developed for N-gene showed an LOD and E-strip sensitivity of log 1.79 ± 0.427 pgµL−1 and log 0.067 µA/pg µL−1/mm2, respectively. The developed assay systems were validated with the clinical samples from COVID-19 outbreaks in 2020 and 2021. This multigene target approach can effectively detect emerging COVID-19 variants using combination of various analytical techniques at testing facilities and in point-of-care settings.

RNA-Based Sensor Systems for Affordable Diagnostics in the Age of Pandemics

“…The findings demonstrated that although the multigene approach enabled positive detection utilizing more target areas, the lateral flow dip strips produced false-negative results for two samples. The designed assays, such as the electrochemical sensor for N-gene and lateral flow device (LFD)-based RT-LAMP test, showed agreement with RT-PCR findings, suggesting their potential for a precise and sensitive diagnosis of COVID-19, particularly in the setting of developing variants with numerous mutations. − …”

“…The designed assays, such as the electrochemical sensor for N-gene and lateral flow device (LFD)-based RT-LAMP test, showed agreement with RT-PCR findings, suggesting their potential for a precise and sensitive diagnosis of COVID-19, particularly in the setting of developing variants with numerous mutations. 100 − 102 …”

Laboratory Evaluation of a SARS-CoV-2 RT-LAMP Test

“…This can be circumvented by visualising the amplification results under the illumination of UV light, which can be observed by the naked eye [ 18 ]. A slightly more sophisticated read-out method is the use of lateral flow-based strips for the detection of amplified SARS-CoV-2 viral mRNA [ 19 , 20 ]. A further simplification could be sought by using less sophisticated (or even non) nucleic acid extraction methods [ 21 ], but these will need further validation, particularly for RNA.…”

Different forms of loop-mediated isothermal amplification (LAMP) for the detection of human viruses

“…For instance, a RT-LAMP assay, combined with electrochemical biosensors, has been employed for the diagnosis of COVID-19 (SULEMAN, et al 2021;KUMAR et al, 2022b). Briefly, this technology consists of adding amplicons from RT-LAMP to the biosensor surface with the aim of improving the detection of virus target sequences through hybridization with specific probes (SAXENA et al 2022). Additionally, RT-LAMP, combined with CRISPR/Cas, has been employed to improve the sensitivity and specificity of SARS-COV-2 detection (SELVAM et al, 2021;REZAEI et al, 2021;BHATT et al 2022).…”