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.

Fig. 1

Phylogeny of the nidovirus RdRp domain and the target RdRp region. Phylogenetic analyses included five arteri-, two toro-, one bafini- and 19 coronaviruses. The RdRp tree was inferred using maximum likelihood and midpoint pseudo-rooted. Tip labels include accession numbers and virus acronyms. Viruses used to construct primers are highlighted in bold and those used to test the described primers are marked with asterisks. Alpha-, Beta- and Gammacoronavirus genera are indicated by vertical bars and symbols. The inset (grey background) shows a neighbor-joining tree based on the ∼150 nt region flanked by the A/B primer pair sets. Support for internal nodes of at least 80 out of 100 non-parametric bootstraps is indicated by numbers. The scale bars represent the average numbers of substitutions per sequence position.

Fig. 2

Design of corona/torovirus primers. From top to bottom shown are amino acid and nucleotide alignments for 11 coronaviruses and one torovirus with derived primers following. The amino acid sequence alignments are given for the two most conserved RdRp regions including motifs A and B. The parts of the amino acid alignments (depicted with black arrows) are also shown as nucleotide alignments. Virus names, the genome accession numbers and their grouping into phylogenetic clusters (corresponding to genera) may be indicated. The cluster numbers 1–4 represent the genera of the respective virus species: 1, alphacoronaviruses; 2, betacoronaviruses; 3, gammacoronaviruses; and 4, torovirus. The nucleotide alignments were used to generate three pairs of primers (color coded), original CT12-oCODEHOP, in-silico CT12-sCODEHOP and modified CT12-mCODEHOP. The primer degeneracy (d), the number of the individual primer-virus mismatches (mismatch) and the average number of mismatches per primer (average) are indicated using the color code of three primer sets (see above). A vertical dotted line separates the consensus clamp and the degenerate portion (underlined) of the primers, which are defined according to CODEHOP, and the respective portions of the nucleotide alignments. Red arrows indicate the four positions within the consensus clamp of the CT12-mCODEHOP primers (bold font), that have been made either degenerate (D) or replaced with inosine (I). Note that there are also other positions in the clamp that deviate between CT12-mCODEHOP and CT12-oCODEHOP.

Fig. 3

Validation CT12-mCODEHOP primers on total RNA isolated from coronavirus infected cells: two-step RT-PCR assay. The sensitivity of the assay was evaluated on 10-fold dilution series of quantified RNA templates obtained from cells infected with four coronaviruses and one torovirus. Shown are agarose gels electrophoresis with a 1 kb DNA molecular marker, virus-specific amplification bands of ∼200 bp and primer dimer bands.

Fig. 4

Validation CT12-mCODEHOP primers on total RNA isolated from coronavirus infected cells: one-step RT-PCR assay. The sensitivity of the assay was evaluated on 10-fold dilution series of quantified coronavirus RNA templates obtained from cells infected with four coronaviruses. Shown are portions of agarose gels electrophoresis images with virus-specific bands.

Fig. 5

Comparative evaluation of the sensitivity of the CT12-mCODEHOP and CT12-oCODEHOP primer sets toward copy virus RNA templates. The performance of the two primer sets was compared using the one-step RT-PCR assay, and 10-fold dilution series of quantified cRNA templates produced for 5 coronaviruses and one torovirus. Additionally, the effect of the presence of 10¹⁰ copies of cellular RNA (bRNA) per reaction on the performance of the CT12-mCODEHOP primer set was analyzed. Shown are portions of agarose gel electrophoresis images with virus-specific bands.

Fig. 6

Detection of two bovine torovirus field isolates. Agarose gel electrophoresis of the BCoV-specific products from the one-step RT-PCR assay that was directed by CT12-mCODEHOP primers. The BCoV identity of the products was confirmed by sequencing analysis.