Real-Time Reverse Transcription-PCR Assay Panel for Middle East Respiratory Syndrome Coronavirus (original) (raw)
Related papers
PLoS Currents, 2013
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.
Iranian Red Crescent Medical Journal, 2016
Background: Coronaviruses (CoVs) are large ribonucleic acid (RNA) viruses causing primarily respiratory disease in humans. A novel human coronavirus, subsequently named middle east respiratory syndrome coronavirus (MERS-CoV), was first reported in Saudi Arabia in September of 2012. With increasing numbers of infections and deaths from MERS-CoV, development of a rapid and reliable kit was crucial to prevent further spread of MERS-CoV. Objectives: In this study, we present two real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays for in-house rapid and sensitive diagnostic testing of MERS-CoV, detecting the regions upstream of the envelope gene (upE) and open reading frame (ORF) 1b, respectively, for initial screening and final confirmation of MERS-CoV infection, as recommended by the world health organization (WHO). Materials and Methods: In this experimental study, acquiring patient samples was difficult; thus, according to WHO recommendations and standard protocols, we synthesized RNA sequences of upE and ORF1b genes as the template signatures and TaqMan based-diagnostic rRT-PCR assays were carried out using these synthetic genes for detection of MERS-CoV. In this research, we also inaugurated a cell-free system to transcribe these RNA sequences using the DNA templates synthesized. Results: The upE and ORF1b based one-step rRT-PCR assays were optimized by testing several times via different synthetic RNAs, and validation results were highly successful. The sensitivity obtained for upE was fewer than ten copies of RNA template per reaction and for ORF1b was 50 or fewer copies per reaction. Conclusions: This study showed that the developed rRT-PCR assays are rapid, reliable, reproducible, specific, sensitive, and simple tools for detection of MERS-CoV. Finally, a kit consisting of two assay signatures and controls was assembled, which can be distributed to public health laboratories in Iran to support international MERS-CoV surveillance and public health response.
Middle East Respiratory Syndrome Coronavirus (MERS-CoV): Announcement of the Coronavirus Study Group
Journal of Virology, 2013
A countrywide survey in Oman revealed Middle East respiratory syndrome coronavirus (MERS-CoV) nucleic acid in five of 76 dromedary camels. Camel-derived MERS-CoV sequences (3,754 nucleotides assembled from partial sequences of the open reading frame (ORF)1a, spike, and ORF4b genes) from Oman and Qatar were slightly different from each other, but closely related to human MERS-CoV sequences from the same geographical areas, suggesting local zoonotic transmission. High viral loads in nasal and conjunctival swabs suggest possible transmission by the respiratory route.
International Journal of Infectious Diseases, 2019
The identification and analysis of viral etiological agents from suspected Middle East respiratory syndrome coronavirus (MERS-CoV) cases admitted to Prof. Dr. Sulianti Saroso Infectious Disease Hospital (IDH) using molecular assays. Methods: Biological samples were collected from 13 hospitalized patients suspected of MERS-CoV infection in Prof. Dr. Sulianti Saroso IDH from July 2015 to December 2016. The majority of patients presented with pneumonia, with symptoms including fever (!37.5 C), labored breathing, and cough, and with a history of travel to the Middle East. Viral RNA was isolated and converted to cDNA, which was used as a template for the detection of 12 viral panels using conventional PCR and sequencing. Results: Viral etiological agents detected in the patients were enterovirus D68, dengue virus type 3, rhinovirus C, human coronavirus 229E, herpes simplex virus type 1, influenza virus H1N1, influenza virus H3N2, human metapneumovirus, and rhinovirus A60. Conclusions: The sequences of nine viral agents under different taxa were detected in suspected MERS-CoV patients, including influenza virus, paramyxovirus, coronavirus, enterovirus, human metapneumovirus, and herpesvirus.
Journal of Clinical Microbiology, 2004
First-generation reverse transcription-PCR (RT-PCR) assays for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) gave false-negative results in a considerable fraction of patients. In the present study, we evaluated two second-generation, replicase (R) gene-based, real-time RT-PCR test kits-the RealArt HPA coronavirus LC kit (Artus, Hamburg, Germany) and the LightCycler SARS-CoV quantification kit (Roche, Penzberg, Germany)-and a real-time RT-PCR assay for the nucleocapsid (N) gene. Detecting the N-gene RNA might be advantageous due to its high abundance in cells. The kits achieved sensitivities of 70.8% (Artus) and 67.1% (Roche) in 66 specimens from patients with confirmed SARS (samples primarily from the upper and lower respiratory tract and stool). The sensitivity of the N-gene assay was 74.2%. The differences in all of the sensitivities were not statistically significant (P ؍ 0.680 [analysis of variance]). Culture cells initially contained five times more N-than R-gene RNA, but the respective levels converged during 4 days of virus replication. In clinical samples the median concentrations of R-and N-gene RNA, respectively, were 1.2 ؋ 10 6 and 2.8 ؋ 10 6 copies/ml (sputum and endotracheal aspirates), 4.3 ؋ 10 4 and 5.5 ؋ 10 4 copies/ml (stool), and 5.5 ؋ 10 2 and 5.2 ؋ 10 2 copies/sample (throat swabs and saliva). Differences between the samples types were significant but not between the types of target RNA. All (n ؍ 12) samples from the lower respiratory tract tested positive in all tests. In conclusion, the novel assays are more sensitive than the first-generation tests, but they still do not allow a comprehensive ruling out of SARS. Methods for the routine sampling of sputum without infection risk are needed to improve SARS RT-PCR.
Journal of Medical Virology, 2018
The first reported case of Middle East respiratory syndrome coronavirus (MERS-CoV) infection was identified in Saudi Arabia in September 2012, since which time there have been over 2000 laboratory-confirmed cases, including 750 deaths in 27 countries. Nucleic acid testing (NAT) is the preferred method for the detection of MERS-CoV. A single round of a Proficiency Testing Program (PTP) was used to assess the capability of laboratories globally to accurately detect the presence of MERS-CoV using NAT. A panel of eleven lyophilized specimens containing different viral loads of MERS-CoV, common coronaviruses, and in vitro RNA transcripts was distributed to laboratories in all six World Health Organization regions. A total of 96 laboratories from 79 countries participating in the PTP, with 76 of 96 (79.2%) reporting correct MERS-CoV results for all nine scored specimens. A further 10 laboratories (10.4%) scored correctly in eight of nine specimens of the PTP. The majority of laboratories demonstrated satisfactory performance in detecting the presence of MERS-CoV using NAT. However, some laboratories require improved assay sensitivity, reduced cross contamination of samples, and improved speciation of coronavirus subtypes for potentially complex clinical specimens. Further PTP and enhanced links with expert laboratories globally may improve the laboratory performance.
Transactions of The Royal Society of Tropical Medicine and Hygiene, 2021
Background There have been 2562 laboratory-confirmed cases of Middle East respiratory syndrome coronavirus (MERS-CoV) in 27 countries, with a case fatality rate of 34.5%. Data on the turnaround time (TAT) are lacking. We report TAT for MERS-CoV samples over time. Methods This is a monocentric study and the TAT for the reporting of 2664 MERS-CoV polymerase chain reaction (PCR) results were calculated in hours from the time of the receipt of respiratory samples to the reporting of the results. Results The mean TAT±standard deviation was significantly lower in 2018 compared with previous years (19.25±13.8). The percentage of samples processed within 24 h increased from 42.3% to 73.8% in 2015 and 2018, respectively (p<0.0001). The mean TAT was 19.2 h in 2018 and was significantly lower than previous years. Conclusions The TAT for the MERS-CoV results decreased during the study period. Timely reporting of MERS-CoV PCR results may aid in further enhancing infection control measures.