Development and Validation of a Rapid Immunochromatographic Assay for Detection of Middle East Respiratory Syndrome Coronavirus Antigen in Dromedary Camels

Abstract

We present here a rapid immunochromatographic assay for the detection of Middle East respiratory syndrome coronavirus (MERS-CoV) antigen in the nasal swabs of dromedary camels. The assay is based on the detection of MERS-CoV nucleocapsid protein in a short time frame using highly selective monoclonal antibodies at room temperature. The relative sensitivity and specificity of the assay were found to be 93.90% and 100%, respectively, compared to that of the UpE and open reading frame 1A (Orf1A) real-time reverse transcriptase PCR (RT-PCR). The results suggest that the assay developed here is a useful tool for the rapid diagnosis and epidemiological surveillance of MERS-CoV infection in dromedary camels. Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly identified human coronavirus associated with severe pulmonary syndrome and renal failure in infected patients (1). To date, a total of 843 persons in 21 different countries have been infected by the virus, with a resulting 37.95% mortality rate (2). The current MERS-CoV outbreak investigations suggest that camels are a source of human infections. Nevertheless, the exact route of transmission from camels to humans remains unclear (3).MERS-CoV is primarily diagnosed using molecular techniques. These include real-time reverse transcriptase PCR (RT-PCR) (4, 5), reverse transcription-loop-mediated isothermal amplification (RT-LAMP) (6) and reverse transcription-recombinase polymerase amplification (RT-RTPA) (7). Moreover, several serological assays have been used to detect MERS-CoV or closely related viruses in seropositive camels. These are protein microarrays (8-10), a recombinant spike immunofluorescent assay (11, 12), indirect enzyme-linked immunosorbent assay (ELISA) (13), microneutralization, and spike pseudoparticle neutralization (14). However, none of the serological tests have provided proof of the precise presence of MERS-CoV in camels.Molecular tests are relatively expensive, not available in all laboratories, and are mainly used for confirmatory purposes. For the purpose of screening of large numbers of animals in a short period of time, molecular tests are considered impractical; therefore, a rapid, cheap, sensitive, and specific test is needed for the diagnosis of MERS-CoV in camels. Here, we report the development and validation of an immunochromatographic assay (ICA) for the rapid qualitative detection of MERS-CoV antigen in dromedary camels. The assay is based on the detection of MERS-CoV nucleocapsid protein by highly selective monoclonal antibodies. MATERIALS AND METHODSThis study was carried out in two phases during the period of August to October 2014. In the first phase, the ICA was developed at the BioNote laboratory (South Korea). In the second phase, the performance and validation of the ICA were carried out at the veterinary laboratories of the Abu Dhabi Food Control Authority (United Arab Emirates).Peptides and monoclonal antibody synthesis. At first, the hydrophilic regions of the nucleocapsid gene of MERS-CoV were analyzed b...

The prevalence of Middle East respiratory Syndrome coronavirus (MERS-CoV) infection in livestock and temporal relation to locations and seasons

“…Also they reported that the ICA was less sensitive for the detection of MERS-CoV antigen (10 5 TCID50) than was the UpE real-time PCR (10 4 TCID50). The difference between the assay sensitivities might be regarded to the release of subgenomic RNA after the onset of cytopathogenic effect (CPE) in cell culture, including the UpE target fragment, as previously reported [13,14,19]. Also the Rapid MERS-CoV Ag assay cannot detect MERS-CoV loads < 10 4 or Ct value >30.…”

Coronaviruses — drug discovery and therapeutic options

“…Point-of-care nucleic amplification tests such as RT-loop-mediated isothermal amplification and RT-isothermal recombinase polymerase amplification are suitable for field evaluation, especially in resource-limited areas 250,251 . Similarly, assays that detect abundantly expressed CoV antigens, such as the nucleocapsid protein, can be used for fast and high-throughput laboratory diagnosis without requiring biosafety level 3 containment 252,253 . The rapid availability of complete genome sequences of most human and animal CoVs has minimized the time required for the design of new RT-PCR assays, source identification and molecular surveillance for emerging CoVs 254 .…”

MERS: Progress on the global response, remaining challenges and the way forward

“…The nonspecific, and sometimes unusual, clinical presentation of MERS in humans, makes early diagnosis difficult in health care facilities. While several highly specific and sensitive molecular and serologic assays exist for diagnosis in animals and humans Corman et al, 2012aCorman et al, , 2012bLu et al, 2014;Perera et al, 2013a;Reusken et al, 2013b;Müller et al, 2015;Song et al, 2015), there was a clear call from representatives from affected countries for the development of a rapid diagnostic test to improve identification and isolation of primary human cases in health care facilities. A full landscape analysis of MERS-CoV diagnostics will be published separately (Van Kerkhove, personal communication).…”