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

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.

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.

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

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.