A Novel Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification Detection Platform: Application to Diagnosis of COVID-19.

Yi Wang,Ting Chen,Sha Li,Jun Tai,Huan Li,Xiaoying Fu,Limei Han,Licheng Wang,Hai Chen,Xiaoxia Wang,Xiong Zhu,Zhengkun Li,Shaojin Chen,Yuanli Li,Mei Xing

doi:10.3389/fbioe.2021.748746

Abstract

The ongoing Corona virus disease (COVID-19) outbreak has become a huge global health concern. Here, we reported a novel detection platform based on the loop-mediated isothermal amplification (LAMP), termed real-time reverse transcription LAMP (rRT-LAMP) and applied it for the diagnosis of COVID-19 (COVID-19 rRT-LAMP). rRT-LAMP integrates reverse transcription, LAMP amplification, restriction endonuclease cleavage and real-time fluorescence detection into one-pot reaction, and facilitates the diagnosis of COVID-19 at 64°C for only 35 min. The ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2 were detected for diagnosing COVID-19. The limit of detection (LoD) of COVID-19 rRT-LAMP assay was 14 copies (for each marker) per vessel, and no positive results were obtained from non-SARS-CoV-2 templates. To demonstrate its feasibility, a total of 33 oropharynx swab samples collected from COVID-19 patients also were diagnosed as SARS-CoV-2 infection using COVID-19 rRT-LAMP protocol. No cross-reactivity was yielded from 41 oropharynx swab samples collected from non-COVID-19 patients. These data suggesting that the COVID-19 rRT-LAMP assay is a potential detection tool for the diagnosis of SARS-CoV-2 infection in clinical, field and disease control laboratories, and will be valuable for controlling the COVID-19 epidemic.

Highlights

The ongoing COVID-19 (Corona virus disease) epidemic caused by SARS-CoV-2 that had previously not been documented in animals or humans, has become a major global public health concern (Huang et al, 2020)
Polymerase chain reaction (PCR)-based methodologies, including real-time PCR (RT-PCR) and real-time reverse transcription PCR are characterized by rapid detection, high specificity and sensitivity, which have been employed for diagnosis of COVID-19 (Carter et al, 2020)
We reported a novel mode of RT-loop-mediated isothermal amplification (LAMP), termed realtime reverse transcription LAMP, which was employed for diagnosing COVID-19 (COVID-19 rRT-LAMP)

Summary

Introduction

The ongoing COVID-19 (Corona virus disease) epidemic caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) that had previously not been documented in animals or humans, has become a major global public health concern (Huang et al, 2020). Given the rapid spread speed (R0 3.28) and mortality rate (2.3%) of SARS-CoV-2 infection, the valuable diagnostic tools are urgently required for rapidly screening suspected cases, accurately diagnosing COVID-19 and performing epidemiological surveillance. The detection of SARS-CoV-2 RNA has been approved to be useful for the diagnosis of COVID-19, which was beneficial to preventing the spreading, controlling the sources of infection and helping patients to prevent the disease progression (Li et al, 2020; Shen et al, 2020). At the early stage of COVID-19 epidemic, the next-generation sequencing was employed for detecting SARS-CoV-2 RNA in various clinical specimens, while it was not available in field and clinic settings due to its longer sequencing time and high needs for equipment (Wu et al, 2020b; Zhu et al, 2021). Further development of simpler, more rapid and sensitive detection tools to diagnose COVID-19 are still needed

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Discussion

Conclusion