development of a simple, fast and portable recombinase aided amplification assay for 2019-ncov

Conditions: New Coronavirus

Summary: 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.

Eligibility criteria:
inclusion criteria: * 1. suspected cases (formerly observed cases) meet the following 2 at the same time: epidemiological history there was a history of travel or residence in wuhan within two weeks before the onset of illness; or patients who had had fever from wuhan with respiratory symptoms within 14 days before the onset of illness, or had clustered onset. clinical manifestations 1. fever; 2. it has the imaging characteristics of pneumonia mentioned above; 3. the total number of white blood cells is normal or decreased, or the lymphocyte count is decreased in the early stage of onset. * 2. confirmed cases on the basis of meeting the criteria for suspected cases, sputum, throat swabs, lower respiratory tract secretions, and other specimens were tested by real-time fluorescent rt-pcr for positive nucleic acid detection of new coronavirus; or viral gene sequencing was highly homologous with known new coronaviruses. exclusion criteria: * 1. influenza virus, parainfluenza virus, adenovirus, respiratory syncytial virus, rhinovirus, human metapneumovirus, sars coronavirus, and other known other viral pneumonia; * 2. mycoplasma pneumoniae, chlamydia pneumonia, and bacterial pneumonia; non-infectious diseases such as vasculitis, dermatomyositis, and organizing pneumonia.

Keywords: recombinase aided amplification assay 2019-ncov diagnosis specificity sensitivity