Development of a Simple, Fast and Portable Recombinase Aided Amplification Assay for 2019-nCoV

Overview

In late December 2019, several local health facilities reported clusters of patients with pneumonia of unknown cause that were epidemiologically linked to a seafood and wet animal wholesale market in Wuhan, Hubei Province, China. It is now confirmed that the etiology of this outbreak is a novel coronavirus, namely, 2019-nCoV. Of critical importance is rapid and simple diagnostic method to be used in clinical settings to timely inform and refine strategies that can prevent, control, and stop the spread of 2019-nCoV. Recombinase aided amplification (RAA) assay is a novel isothermal nucleic acid amplification technique in recent years, which has a variety of the advantages including high specificity and sensitivity, rapid detection (30 min), low cost, low equipment requirements and simple operation. The has successfully detected a variety of pathogens using this technique. To develop a RAA assay for 2019-nCoV with the advantages of high speed, simple operation and low cost, and overcomes the shortcomings of the existing molecular detection methods. The investigators established a real time reverse-transcription RAA (RT-RAA) assay for detection of 2019-nCoV. This assay was performed at 42°C within 30min using a portable real-time fluorescence detector, Recombinant plasmids containing conserved ORF1ab genes was used to analyze the specificity and sensitivity. Clinical specimens from patients who were suspected of being infected with 2019-nCoV were used to evaluate the performance of the assay. In parallel, The investigators also used the commercial RT-qPCR assay kit for 2019-nCoV as a reference.

In late December 2019, several local health facilities reported clusters of patients with pneumonia of unknown cause that were epidemiologically linked to a seafood and wet animal wholesale market in Wuhan, Hubei Province, China. It is now confirmed that the etiology of this outbreak is a novel coronavirus, namely, 2019-nCoV. Of critical importance is rapid and simple diagnostic method to be used in clinical settings to timely inform and refine strategies that can prevent, control, and stop the spread of 2019-nCoV. Recombinase aided amplification (RAA) assay is a novel isothermal nucleic acid amplification technique in recent years, which has a variety of the advantages including high specificity and sensitivity, rapid detection (30 min), low cost, low equipment requirements and simple operation. The investigators has successfully detected a variety of pathogens using this technique. To develop a RAA assay for 2019-nCoV with the advantages of high speed, simple operation and low cost, and overcomes the shortcomings of the existing molecular detection methods. The investigators established a real time reverse-transcription RAA (RT-RAA) assay for detection of 2019-nCoV. This assay was performed at 42°C within 30min using a portable real-time fluorescence detector, Recombinant plasmids containing conserved ORF1ab genes was used to analyze the specificity and sensitivity. Clinical specimens from patients who were suspected of being infected with 2019-nCoV were used to evaluate the performance of the assay. In parallel, The investigators also used the commercial RT-qPCR assay kit for 2019-nCoV as a reference. Patients who were suspected of being infected with 2019-nCoV in the hospital.