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

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

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.

Study Overview

• Status: Unknown
• Conditions: New Coronavirus
• Intervention / Treatment: Diagnostic test: Recombinase aided amplification (RAA) assay

Detailed Description

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.

• Study Type: Observational
• Enrollment (Anticipated): 50

Contacts and Locations

• Study Contact: Name: Yao Xie, Doctor; Phone Number: 2489 8610-84322200; Email: xieyao00120184@sina.com
• Study Contact Backup: Name: Xuejun Ma, phD; Phone Number: +86 10 58900810; Email: maxj2004@aliyun.com

Study Locations

• China
  • Beijing
    • Beijing, Beijing, China, 100015
      • Recruiting
      • Department of Hepatology Division 2, Beijing Ditan Hospital
      • Contact: Yao Xie, Doctor; Phone Number: 2489 8610-84322200; Email: xieyao00120184@sina.com
      • Contact: Xuejun Ma, phD; Phone Number: +86 10 58900810; Email: maxj2004@aliyun.com

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 3 months to 88 years (Child, Adult, Older Adult)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

• Sampling Method: Probability Sample

Study Population

Patients who were suspected of being infected with 2019-nCoV in the hospital.

Description

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.

Study Plan

How is the study designed?

Design Details

• Observational Models: Cohort
• Time Perspectives: Prospective

Number of groups / cohorts

1

Cohorts and Interventions

Group / Cohort

Intervention / Treatment

RAA assay for 2019-nCoV; a simple, fast and portable recombinase aided amplification (RAA) assay for 2019-nCoV

Diagnostic test: Recombinase aided amplification (RAA) assay; We 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, we also used the commercial RT-qPCR assay kit for 2019-nCoV as a reference. Sample types include either of nasal swab, oral swab, bronchoalveolar-lavage fluid, urea, blood, fecal.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Detection sensitivity is greater than 95%	Detection sensitivity is greater than 95%	at baseline
Detection specificity is greater than 95%	Detection specificity is greater than 95%	at baseline

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Consistent with existing universal reagent detection rates greater than 95%	Consistent with existing universal reagent detection rates greater than 95%	at baseline

Collaborators and Investigators

• Sponsor: Beijing Ditan Hospital
• Investigators: Principal Investigator: Yao Xie, Doctor, Department of Hepatology, Division 2, Beijing Ditan Hospital

Study record dates

Study Major Dates

• Study Start (Actual): January 1, 2020
• Primary Completion (Anticipated): December 31, 2020
• Study Completion (Anticipated): December 31, 2020

Study Registration Dates

• First Submitted: January 26, 2020
• First Submitted That Met QC Criteria: January 26, 2020
• First Posted (Actual): January 29, 2020

Study Record Updates

• Last Update Posted (Actual): April 10, 2020
• Last Update Submitted That Met QC Criteria: April 9, 2020
• Last Verified: April 1, 2020

More Information

Terms related to this study

• Keywords: diagnosis; 2019-nCoV; Sensitivity; Specificity; recombinase aided amplification assay
• Additional Relevant MeSH Terms: Coronaviridae Infections; Nidovirales Infections; RNA Virus Infections; Virus Diseases; Infections; Coronavirus Infections
• Other Study ID Numbers: DTXY022

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No