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. 2013 Dec 12:5:ecurrents.outbreaks.62df1c7c75ffc96cd59034531e2e8364.
doi: 10.1371/currents.outbreaks.62df1c7c75ffc96cd59034531e2e8364.

Reverse transcription recombinase polymerase amplification assay for the detection of middle East respiratory syndrome coronavirus

Ahmed Abd El Wahed  1 , Pranav Patel  2 , Doris Heidenreich  3 , Frank T Hufert  3 , Manfred Weidmann  3
Affiliations

Reverse transcription recombinase polymerase amplification assay for the detection of middle East respiratory syndrome coronavirus

Ahmed Abd El Wahed et al. PLoS Curr. .

Abstract

The emergence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in the eastern Mediterranean and imported cases to Europe has alerted public health authorities. Currently, detection of MERS-CoV in patient samples is done by real-time RT-PCR. Samples collected from suspected cases are sent to highly-equipped centralized laboratories for screening. A rapid point-of-care test is needed to allow more widespread mobile detection of the virus directly from patient material. In this study, we describe the development of a reverse transcription isothermal Recombinase Polymerase Amplification (RT-RPA) assay for the identification of MERS-CoV. A partial nucleocapsid gene RNA molecular standard of MERS-coronavirus was used to determine the assay sensitivity. The isothermal (42°C) MERS-CoV RT-RPA was as sensitive as real-time RT-PCR (10 RNA molecules), rapid (3-7 minutes) and mobile (using tubescanner weighing 1kg). The MERS-CoV RT-RPA showed cross-detection neither of any of the RNAs of several coronaviruses and respiratory viruses affecting humans nor of the human genome. The developed isothermal real-time RT-RPA is ideal for rapid mobile molecular MERS-CoV monitoring in acute patients and may also facilitate the search for the animal reservoir of MERS-CoV.

Keywords: MERS-coronavirus; RPA; point-of-care assay.

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Figures

MERS-CoV RT-RPA assay.
MERS-CoV RT-RPA assay.
Over time development of fluorescence using a dilution range of 107-101 molecules/µl of the RNA molecular standard (Tubescanner Studio Software, Qiagen, Germany). MERS-CoV RT-RPA assay sensitivity is 10 copies and yielded results in maximum 7 minutes. 107 represented by black line; 106, gray; 105, red; 104, blue; 103, green; 102, cyan; 101, dark khaki; negative control, orange. No fluorescence signals was measured for one minute (after 3 minutes of the start of the reaction) because of the need of mixing the content.
Analytical sensitivity of MERS-CoV NC real-time RT-PCR and RT-RPA.
Analytical sensitivity of MERS-CoV NC real-time RT-PCR and RT-RPA.
The analytical sensitivity was determined on RNA molecular standard (8 runs) for real-time RT-PCR (A) and real-time RT-RPA (B). Both assays have a sensitivity of 10 RNA molecules. The RT-RPA assay was much faster than the RT-PCR as the run time of the RT-RPA is between 3-7 minutes for 107 and 101molecules, respectively. While the RT-PCR needed up to 2 hours. Consequently, RT-PCR results is linear, while RT-RPA not.
Probit regression analysis of MERS-CoV RT-RPA assay
Probit regression analysis of MERS-CoV RT-RPA assay
The probit analysis was performed using Statistica software on data of the eight runs of 107-101 RNA molecular standard. The limit of detection at 95% probability (21 RNA molecules) is depicted by a Rhomboid.
See this image and copyright information in PMC

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Grants and funding

The study was funded by the Federal Ministry of Education and Research (BMBF) (project name: RESCheck, ID: FKN:16SV5436).