Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections

Abstract

We present a rigorously validated and 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, targeting the RdRp gene (RdRpSeq assay) and N gene (NSeq assay), where an insertion/deletion polymorphism might exist among different hCoV-EMC strains. Finally, a simplified and biologically safe protocol for detection of antibody response by immunofluorescence microscopy was developed using convalescent patient serum.

Specific serology for emerging human coronaviruses by protein microarray

“…However, even in those eighteen countries it does not follow that there is the capacity for large scale or rapid testing with what are non-commercial and technically demanding tests. Additional molecular assays for sensitive and specific case confirmation have been described [18]. Serological tests have been developed but they are labour-intensive and although they are starting to be used, are not yet suitable for use outside of a few highly specialised laboratories.…”

“…For this reason, it is not recommended to screen asymptomatic patients by simple serological tests such as immunofluorescence, if the possibility is not given to conduct immediate second-stage serology. More information about diagnostic procedures can be found in the following articles [19,18,15,13] and on the University of Bonn website.…”

Critically Ill Patients with Middle East Respiratory Syndrome Coronavirus Infection

“…Diagnosis is, therefore, based on molecular testing by real-time reverse-transcription polymerase chain reaction (rRT-PCR) for two sites in the virus genome: the upstream E protein (upE) for screening and the open reading frame (ORF) 1a or 1b for confirmation [28]. Lower respiratory tract specimens have a higher sensitivity than upper respiratory tract specimens for detecting MERS-CoV [29].…”

Review on Middle East Respiratory Syndrome

“…For RT-rtPCR for sequencing in the N gene the assay employed the same conditions as the RdRpSeq assay, except that the primer sequences were NSeq-Fwd (CCT TCG GTA CAG TGG AGC CA) and NSeq-Rev (GAT GGG GTT GCC AAA CAC AAA C) for the first round and NSeq-Fwd (TGA CCC AAA GAA TCC CAA CTA C) and NSeq-Rev (the same as in the first round) for the second round. The second round will only performed if no product is visible by agarose gel electrophoresis after the first round [36]. The assay for the upE target is considered highly sensitive and is recommended for screening, the ORF1a assay considered of equal sensitivity and the ORF 1b assay less sensitive than the ORF1a assay are used for confirmation.…”