Broadly targeted multiprobe QPCR for detection of coronaviruses: Coronavirus is common among mallard ducks (Anas platyrhynchos)

Abstract

Coronaviruses (CoVs) can cause trivial or fatal disease in humans and in animals. Detection methods for a wide range of CoVs are needed, to understand viral evolution, host range, transmission and maintenance in reservoirs. A new concept, "Multiprobe QPCR", which uses a balanced mixture of competing discrete non- or moderately degenerated nuclease degradable (TaqMan) probes was employed. It provides a broadly targeted and rational single tube real-time reverse transcription PCR ("NQPCR") for the generic detection and discovery of CoV. Degenerate primers, previously published, and the new probes, were from a conserved stretch of open reading frame 1b, encoding the replicase. This multiprobe design reduced the degree of probe degeneration, which otherwise decreases the sensitivity, and allowed a preliminary classification of the amplified sequence directly from the QPCR trace. The split probe strategy allowed detection of down to 10 viral nucleic acid equivalents of CoV from all known CoV groups. Evaluation was with reference CoV strains, synthetic targets, human respiratory samples and avian fecal samples. Infectious-Bronchitis-Virus (IBV)-related variants were found in 7 of 35 sample pools, from 100 wild mallards (Anas platyrhynchos). Ducks may spread and harbour CoVs. NQPCR can detect a wide range of CoVs, as illustrated for humans and ducks.

Isolation and molecular characterization of Sul/01/09 avian infectious bronchitis virus, indicates the emergence of a new genotype in the Middle East

“…Further investigation is necessary to exclude the existence of Iranian isolates in other parts of Iraq. Since diverse coronaviruses have been detected recently in wild birds (Hughes et al, 2009) and it is common among mallard ducks (Muradrasoli et al, 2009), it is very likely that such identical isolates transmitted between Iraq, Israel and Egypt through wild migrated birds, but not through poultry product trade, which does not existing between these countries. Further investigation of IBV in migrated wild birds is necessary to elucidate this measure.…”

Design and validation of consensus-degenerate hybrid oligonucleotide primers for broad and sensitive detection of corona- and toroviruses

“…Several RT-PCR assays targeting highly conserved regions in the replicase gene have been developed for broad detection of various CoVs. Although primers-based methods have been successful in identifying unknown CoVs (Adachi et al, 2004;Stephensen et al, 1999;Muradrasoli et al, 2009;Escutenaire et al, 2007;Sampath et al, 2005;Vijgen et al, 2008;Tong et al, 2009), extension of this approach beyond the subfamily Coronavirinae has not been reported. This study describes the design and validation of a broadly reactive and sensitive one-step RT-PCR assay that detects representatives of three CoV genera, as well as ToVs from samples of different origins including clinical and field specimens.…”

“…Because of its highly conserved structure and function, the pp1ab nsp12 gene (known also as pol gene) encoding an RdRp domain has been frequently targeted for the design of pancoronavirus primers (Stephensen et al, 1999;Moes et al, 2005;Vijgen et al, 2008). More sensitive SYBR and Taqman probe-based real-time RT-PCR methods targeting this gene have also been reported (Escutenaire et al, 2007;Muradrasoli et al, 2009). All these assays have been developed exclusively for coronavirus detection, thus limiting the potential recognition of more distantly related viruses.…”

Absence of Coronaviruses, Paramyxoviruses, and Influenza A Viruses in Seabirds in the Southwestern Indian Ocean

“…Real-time PCR was performed on MS2-positive samples only, following published protocols optimized for the detection of avian IAV, PMV, and COV (respectively: Spackman et al, 2002;Kim et al, 2008;Muradrasoli et al, 2009). The ABsolute Blue qPCR Low ROX Mix (Thermo Fisher Scientific, Surrey, UK) was used in a final volume of 25 mL containing 5 mL of cDNA; PCRs were carried out in a BioRad CFX96 Touch TM (Bio-Rad, Hercules, California, USA) real-time PCR detection system.…”