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.

No novel coronaviruses identified in a large collection of human nasopharyngeal specimens using family-wide CODEHOP-based primers

“…False-negative HCoV-229E and HCoV-NL63 samples had a lower median interquartile Ct value, 28.3 and 28.9, respectively, in comparison to 32.32 for HCoV-OC43, indicating higher viral loads and a lower sensitivity of the assay towards HCoV-229E and HCoV-NL63. These results are in accordance with our previous findings using quantified copy RNA transcripts and HCoVpositive respiratory specimens [23].…”

“…We have recently described the development and validation of modified CODEHOP-based primers that were designed to recognize distantly related corona-and toroviruses of the family Coronaviridae (CT12-mCODEHOP) in a reverse transcription (RT)-PCR assay [23]. These primers detected four known circulating human coronaviruses and two bovine toroviruses directly from biological specimens.…”

“…For virus discovery, GRACE samples were analyzed blindly in a pool of two as reported previously [23]. Employing a multiplex real-time PCR assay designed to detect HCoV-OC43, -229E and -NL63, 69 out of 1800 nasopharyngeal specimens were found to be positive and were subsequently typed using species-specific monoplex assays ( Table 2).…”

Aetiology of lower respiratory tract infection in adults in primary care: a prospective study in 11 European countries

“…HCoV have recently been identified in small numbers of adults with severe pneumonia [10,21], but is not routinely tested for in adult outpatients with CAP or LRTI. We may have underestimated the prevalence of HCoV as HKU-1 testing was not performed and HKU-1 is generally as prevalent as NL63 and OC43 [22]. Infections due to HMPV are mainly described in long-term care facilities [10].…”

HexaPrime: A novel method for detection of coronaviruses

“…This approach is promising, although high variability of viral sequences coupled with limitations to the level of degeneration of primers may lead to false-negative results if viral species are not identical to reference strains. To overcome this problem, the CODEHOP technique was developed, allowing the design of highly degenerated DNA primers based on more conserved protein sequences (Rose et al, 1998;Zlateva et al, 2011).…”