title: Design and validation of consensus-degenerate hybrid oligonucleotide primers for broad and sensitive detection of corona- and toroviruses.
abstract: The ssRNA+ family Coronaviridae includes two subfamilies prototyped by coronaviruses and toroviruses that cause respiratory and enteric infections. To facilitate the identification of new distantly related members of the family Coronaviridae, we have developed a molecular assay with broad specificity. The consensus-degenerated hybrid oligonucleotide primer (CODEHOP) strategy was modified to design primers targeting the most conserved motifs in the RNA-dependent RNA polymerase locus. They were evaluated initially on RNA templates from virus-infected cells using a two-step RT-PCR protocol that was further advanced to a one-step assay. The sensitivity of the assay ranged from 10(2) to 10(6) and from 10(5) to 10(9) RNA copy numbers for individual corona-/torovirus templates when tested, respectively, with and without an excess of RNA from human cells. This primer set compared to that designed according to the original CODEHOP rules showed 10-10(3) folds greater sensitivity for 5 of the 6 evaluated corona-/torovirus templates. It detected 57% (32 of 56) of the respiratory specimens positive for 4 human coronaviruses, as well as stool specimens positive for a bovine torovirus. The high sensitivity and broad virus range of this assay makes it suitable for screening biological specimens in search for new viruses of the family Coronaviridae.
authors.affiliation: Department of Medical Microbiology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.
authors.forename: Kalina T
authors.initials: KT
authors.lastname: Zlateva
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Crusio
authors.forename: Andrey M
authors.initials: AM
authors.lastname: Leontovich
authors.forename: Chris
authors.initials: C
authors.lastname: Lauber
authors.forename: Eric
authors.initials: E
authors.lastname: Claas
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Kravchenko
authors.forename: Willy J M
authors.initials: WJ
authors.lastname: Spaan
authors.forename: Alexander E
authors.initials: AE
authors.lastname: Gorbalenya
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D017124:Conserved Sequence; D017934:Coronavirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D018352:Coronavirus Infections / Q000175:diagnosis / Q000821:virology; D017931:DNA Primers / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017003:Torovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D018176:Torovirus Infections / Q000175:diagnosis / Q000821:virology; D014776:Virus Cultivation
chemical_list: D017931:DNA Primers; D012367:RNA, Viral