Comparative Evaluation of 19 Reverse Transcription Loop-Mediated Isothermal Amplification Assays for Detection of SARS-CoV-2

Dong, Yajuan; Wu, Xiuming; Li, Shenwei; Lu, Renfei; Wan, Zhenzhou; Qin, Jianglei; Yu, Guoying; Jin, Xia; Zhang, Chiyu

doi:10.1101/2020.07.22.20159525

Cited by 8 publications

(9 citation statements)

References 26 publications

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“…It is revealed that three samples out of 28 did not match with the results of the commercial kits (Supplementary Table S1 ). In both assays, either commercial or the current one, it is estimated that the Ct score of those negative samples was higher than > 37.01 which is out of the WHO recommendation 36 . Therefore, in the current mCoV-2 assay, they are called as negative.…”

Section: Discussionmentioning

confidence: 88%

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Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N, RdRP and human RP genes

Tombuloğlu

Sabit

Al‐Khallaf

et al. 2022

Sci Rep

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Corona Virus Disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This pandemic has brought the world to a standstill and threatened human lives. Many methods are known to date to detect this virus. Due to their relative sensitivity, polymerase chain reaction (PCR)-based assays are the most frequently applied and considered the gold standard. However, due to the rapid mutation rate of the viral genome and the emergence of new variants, existing protocols need to be updated and improved. Designing a fast and accurate PCR-based assay is of great importance for the early detection of this virus and more efficient control of the spread of this disease. This study describes a fast, reliable, easy-to-use, and high-throughput multiplex SARS-CoV-2 RT-PCR detection method. The assay was designed to detect two viral genes (N and RdRP) and a human gene (RP) simultaneously. The performance and the sensitivity of the assay were tested in 28 SARS-CoV-2 positive samples and compared with commercial kits, which showed 100% positive percent agreement with a limit of detection (LOD) value of 1.40 and 0.81 copies/µL or 35.13 and 20.31 copies/reaction for RdRP and N genes, respectively. The current assay is found accurate, reliable, simple, sensitive, and specific. It can be used as an optimized SARS-CoV-2 diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

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Section: Discussionmentioning

confidence: 88%

“…The RNA concentration was determined by Nanodrop 2000 (Thermo Fisher Scientific, USA) and the copy number was determined according to the following formula 35 , 36 : …”

Section: Methodsmentioning

confidence: 99%

Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N, RdRP and human RP genes

Tombuloğlu

Sabit

Al‐Khallaf

et al. 2022

Sci Rep

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“…3 Because it does not require a thermocycler, the LAMP process lends itself to both scale-up and miniaturization and can be combined with increasingly sophisticated technology and downstream refinements, including sequencing. Numerous reports of LAMP-based assays for SARS-CoV-2 detection in clinical samples are available, [8][9][10] providing information and encouragement for the development of LAMP-based assays for SARS-CoV-2 in raw sewage.…”

Section: Introductionmentioning

confidence: 99%

RT qLAMP—Direct Detection of SARS-CoV-2 in Raw Sewage

Ongerth

Danielson²

2021

J Biomol Tech

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A highly efficient, selective, and sensitive method for analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in raw sewage was developed and tested to illustrate basic characteristics of the procedure. The method uses reverse transcriptase (RT) loop mediated isothermal amplification (LAMP) in a quantitative application, RT qLAMP. The applicability of this procedure to detection of SARS-CoV-2 in clinical samples has been documented in many reports since early 2020. Basic LAMP characteristics depending on the multiple primer design that produce highly selective and sensitive target amplification virtually free of interferences in complex sample media make it ideal for application to target recognition in raw sewage. Three previously described primer sets targeting ORF1a, Eand N-gene regions were selected and tested to define method performance characteristics and performance for SARS-CoV-2 detection in raw sewage samples from a municipal sewage system serving . 600 000, between July and October, 2020. The virus was detected in all samples from each of three independent interceptors near their treatment terminus. Virus quantities varied significantly between samples and between primer targets within samples. Sewage sampling dates corresponded to relatively low COVID-19 incidence rates reported by the local service area health department. The limited number of samples and aggregating downstream sampling locations did not permit resolving concentration differences. The most significant finding was the ability of the RT qLAMP method to detect SARS-CoV-2 in the raw sewage samples directly without preprocessing to isolate or concentrate the virus or to extract and concentrate viral RNA.

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“…One of three isothermal amplification technologies currently available in the United States for at-home detection of SARS-CoV-2 utilizes reverse transcription RPA (RT-RPA), and has been shown to detect the virus in nasal swab specimens with as low as twenty genome copies (31, 32). Given their robust sensitivity and specificity, RT-RPA assays are optimized to detect SARS-CoV-2 during the peak period of transmission in individuals with pre-symptomatic, symptomatic, and/or asymptomatic infections (28, 33, 34).…”

Section: Introductionmentioning

confidence: 99%

Comparative evaluation of rapid isothermal amplification and antigen assays for screening testing of SARS-CoV-2

Salcedo¹,

Sena²,

Qu³

et al. 2021

Preprint

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Human transmission of SARS-CoV-2 and emergent variants of concern has continued to occur globally, despite mass vaccination campaigns. Public health strategies to reduce virus spread should therefore rely, in part, on frequent screening with rapid, inexpensive, and sensitive tests. We evaluated two digitally integrated rapid tests and assessed their performance using stored nasal swab specimens collected from individuals with or without COVID-19. An isothermal amplification assay combined with a lateral flow test had a limit of detection of 10 RNA copies per reaction, and a positive percent agreement (PPA)/negative percent agreement (NPA) during the asymptomatic and symptomatic phases of 100%/100% and 95.83/100%, respectively. Comparatively, an antigen-based lateral flow test, had a limit of detection of 30,000 copies, and a PPA/NPA during the asymptomatic and symptomatic phases of 82.86%/98.68% and 91.67/100%, respectively. Both the isothermal amplification and antigen-based lateral flow tests had optimized detection of SARS-CoV-2 during the peak period of transmission; however, the antigen-based test had reduced sensitivity in clinical samples with qPCR Ct values greater than 29.8. Low-cost, high-throughput screening enabled by isothermal amplification or antigen-based techniques have value for outbreak control.

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Comparative Evaluation of 19 Reverse Transcription Loop-Mediated Isothermal Amplification Assays for Detection of SARS-CoV-2

Cited by 8 publications

References 26 publications

Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N, RdRP and human RP genes

Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N, RdRP and human RP genes

RT qLAMP—Direct Detection of SARS-CoV-2 in Raw Sewage

Comparative evaluation of rapid isothermal amplification and antigen assays for screening testing of SARS-CoV-2

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