Detecting MERS-CoV by PCR and sequencing

We have developed and validated a set of RT-PCR assays for the detection of MERS-CoV. This is the test recommended by WHO. We provide control material necessary for the conduction of the test to laboratories worldwide. Please consult the information below, and please read the Discussion section of the articles for important recommendations.

Corman VM, Eckerle , Bleicker , Zaki A, Landt O, Eschbach-Bludau M, van Boheemen S, Gopal R, Ballhause M, Bestebroer TM, Muth D, Müller MA, Drexler JF, Zambon M, Osterhaus AD, Fouchier RM, Drosten C
Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction.
Euro Surveill. 2012; 17(39)

In this article we present two real-time reverse-transcription polymerase chain reaction assays for MERS-CoV, targeting regions upstream of the E gene (upE) or within open reading frame (ORF)1b (27 September 2012).

Corman VM, Müller MA, Costabel U, Timm J, Binger T, Meyer B, Kreher P, Lattwein E, Eschbach-Bludau M, Nitsche A, Bleicker T, Landt O, Schweiger B, Drexler JF, Osterhaus AD, Haagmans BL, Dittmer U, Bonin F, Wolff T, Drosten C
Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections.
Euro Surveill. 2012; 17(49)

In this second article we present a highly sensitive confirmatory real-time RT-PCR assay (1A assay) that can be used in combination with the previously reported upE assay. Two additional RT-PCR assays for sequencing are described (RdRpSeq assay and NSeq assay). Finally, a protocol for detection of antibody response by immunofluorescence microscopy was developed (06 December 2012).

 

We provide here bench protocols that summarizes the formulation of the MERS-CoV test in a practical way:

Bench protocol (upE and Orf 1b assay)

Bench protocol (1A assay)

Bench protocol (RdRpSeq and NSeq assay)

Sequence alignments (as zipped *.fas files) comparing the results of RdRpSeq and NSeq sequencing assays. The sequences obtained from a patient from Essen, Germany, the HCoV-EMC/2012 and the London sequences are included.

Download here and here.

Positive control

We provide positive control material (for the upE assay and the 1A assay) to qualified laboratories worldwide. The material is a small fragment (< 600 bp) of synthetic RNA representing the part of the viral genome that is detected by the real-time PCR assays we developed. We notify WHO of those entities that have received the material. In order to keep the required administrative work to a manageable level, the ordering and shipping data are handled through the Europan Virus Archive (EVA), a non-commercial entity funded by a European Union infrastructure grant. Please address your request via the EVA homepage. EVA will immediately notify us and we will initiate shipment as soon as possible. The material is shipped from our institution in Bonn/Germany. A small non-profit charge applies for the shipment. Exemptions are possible. Öffnet externen Link im aktuellen FensterMore information

Öffnet externen Link im aktuellen FensterLink for ordering the "upE assay" positive control

Öffnet externen Link im aktuellen FensterLink for ordering the "1A assay" positive control

Download results of both real-time RT-PCRs (upE and 1A assay) on serial dilutions of the corresponding positive controls and the sequences of both controls.

 

Oligonucleotides

Oligos (=primers and probes) as published can be retrieved from stock at Tib-Molbiol, Berlin. The ordering of these reagents from stock provides the advantage of receiving reagents from a large synthesis lot that has been quality-tested on actual virus material in our laboratories, while the oligonucleotides themselves have not physically passed through our facilities. This excludes any possibility of reagent contamination. Öffnet externen Link im aktuellen FensterMore information

 

 

Serological testing for MERS-CoV

Recommendations regarding serological testing

No official recommendations are currently available regarding serological tests. The following recommendations are based on our own experiences and should be regarded as a personal opinion and statement by C. Drosten and M.A. Müller. For more detailed information on serological testing and evaluation please refer to:

Drosten C, Meyer B, Müller MA, Corman VM, Al-Masri M, Hossain R, Madani H, Sieberg A, Bosch BJ, Lattwein E, Alhakeem RF, Assiri AM, Hajomar W, Albarrak AM, Al-Tawfiq JA, Zumla AI, Memish ZA
Transmission of MERS-coronavirus in household contacts.
N. Engl. J. Med. 2014; 371(9):828-35.

Please also read the latest interim WHO recommendation (revised in September 2014).

 

We are currently recommending a two-step diagnostic approach:

Step 1. Recombinant enzyme-linked immunosorbent assay  (ELISA)

Substrate: purified recombinant spike protein (subunit 1 domain) of MERS-CoV

Testing material: Human serum, diluted 1:100

Diagnostic parameter: Immunoglobulin G (Ig)

Alternative to ELISA:  Conventional immunofluorescence assay (cIFA)

Substrate (glass slides): MERS-CoV infected Vero cells mixed 1:1 with non-infected cells (inactivated)

Testing material: Human serum, diluted 1:40 /1:100

Diagnostic parameters: Immunoglobulin G (Ig)G and IgM in patient serum

Step 2. A) Recombinant IFA (rIFA) using spike protein

Substrate (glass slides): Transfected Vero cells expressing recombinant spike protein of MERS-CoV. Remark: For differential rIFA prototype humanpathogenic CoV (HCoV-NL63, -229E, -OC43, -HKU-1, SARS-CoV) should be included.

Testing material: Human serum, diluted 1:40

Diagnostic parameters: Immunoglobulin G (Ig)G and IgM in patient serum

B) Confirmation by virus plaque reduction neutralization test (PRNT)

Substrate: Vero B4 cells and infectious MERS-CoV strain EMC (requires BSL3 laboratory)

Testing material: Heat-inactivated serum/ dilutions starting at 1:10 until 1:1,280

Diagnostic parameter: MERS-CoV neutralizing Ig in patient serum. According to our experience a 50% plaque reduction at a serum dilution of 1:20 can be rated positive.

Important information: The use of the mentioned serological methods is only to be applied in individuals with symptoms according to WHO case definition and a history of proven exposure to a laboratory-confirmed case of MERS-CoV during the infectious period.

We provide rIFA slides in limited quantities to qualified laboratories - please contact us (muller(at)virology-bonn.de). Larger quantities of the recombinant ELISA and conventional IFA slides can also be ordered via EUROIMMUN (contact: Dr. Erik Lattwein, e.lattwein(at)euroimmun.de, EUROIMMUN Lübeck, Germany).

MERS-CoV provides a fine granular cytoplasmatic fluorescence pattern sparing the nucleus. Examples are provided in the paper and in the bench protocol referred below. Any sign of reactivity must be confirmed in consultation with a reference institution before any further steps toward notification are taken.

It is important to know that IFA will generate false-positive results due to cross-reactivity between anti-HCoV antibodies. It is impossible to discriminate cross-reactive antibodies by IFA.

We provide reference services for serology. We re-test reactive sera tested by conventional IFA or other serological assays (e.g. ELISA, Western blot analysis) in differential rIFA and plaque-reduction neutralization tests for confirmation. These highly specific tests are less cross-reactive but cannot be shipped to other laboratories. Please contact us. We will provide rapid assistance wherever possible.

Publication about assays for laboratory confirmation of novel human coronavirus (MERS-CoV) infections, including suggestions for confirmation testing by serology:

Corman VM, Müller MA, Costabel U, Timm J, Binger T, Meyer B, Kreher P, Lattwein E, Eschbach-Bludau M, Nitsche A, Bleicker T, Landt O, Schweiger B, Drexler JF, Osterhaus AD, Haagmans BL, Dittmer U, Bonin F, Wolff T, Drosten C
Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections.
Euro Surveill. 2012; 17(49)

Bench protocol: Conventional immunofluorescence assay (cIFA)

Bench protocol: Recombinant immunofluorescence assay (rIFA)

Our work on detecting and diagnosing emerging viruses is supported by EMPERIE, an EU FP7 project. Öffnet externen Link im aktuellen FensterMore information

Our applied and basic research in related fields is supported by the German Ministry of Education and Research (BMBF), as well as the German National Research Council (DFG). We are a part of the German Centre for Infection Research (DZIF).

Forschungsdatenbank

Für mehr Details nutzen Sie bitte auch die undefinedForschungsdatenbank der Charité.