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. 2004 Dec 1;122(1):29-36.
doi: 10.1016/j.jviromet.2004.07.008.

Comprehensive detection and identification of human coronaviruses, including the SARS-associated coronavirus, with a single RT-PCR assay

D Adachi  1 , G Johnson, R Draker, M Ayers, T Mazzulli, P J Talbot, R Tellier
Affiliations

Comprehensive detection and identification of human coronaviruses, including the SARS-associated coronavirus, with a single RT-PCR assay

D Adachi et al. J Virol Methods. .

Abstract

The SARS-associated human coronavirus (SARS-HCoV) is a newly described, emerging virus conclusively established as the etiologic agent of the severe acute respiratory syndrome (SARS). This study presents a single-tube RT-PCR assay that can detect with high analytical sensitivity the SARS-HCoV, as well as several other coronaviruses including other known human respiratory coronaviruses (HCoV-OC43 and HCoV-229E). Species identification is provided by sequencing the amplicon, although a rapid screening test by restriction enzyme analysis has proved to be very useful for the analysis of samples obtained during the SARS outbreak in Toronto, Canada.

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Figures

Fig. 1
Fig. 1
Alignment calculated from the nucleotide sequence of segments homologous to the segment [15211, 15430] of SARS-HCoV strain TOR2 (GenBank Accession number AY274119) from 12 other species of coronaviruses. HCoV-NL63 (AY567487): human coronavirus NL63. HCoV-229E (NV_002645): human respiratory coronavirus 229E. PEDV (NC_003436): porcine epidemic diarrhea virus. FIPV (AF124987): feline infectious peritonitis virus. CCoV (AF124986): canine coronavirus. TGEV (AJ271965): porcine transmissible gastroenteritis virus. SDAV (AF124990): sialodacryoadenitis virus. MHV (NC_001846): murine hepatitis virus. BCoV (NC_003045): bovine coronavirus. HCoV-OC43 (AF124989): human respiratory coronavirus OC43. IBV (NC_001451): avian infectious bronchitis virus. TCoV (AF124991): turkey coronavirus. Also aligned with the coronaviruses sequences are the sequences of the sense primer (CORO1) and the complement of the antisense primer (CORO2rc). Dots denote homology with the consensus sequence on top. The sequence alignment was calculated using ClustalX for Windows version 1.81 (Thompson et al., 1997) and edited with Genedoc version 2.3 for Windows (Nicholas K.B., 1997).
Fig. 2
Fig. 2
(A) Phylogenetic tree calculated from the nucleotide sequence of segments homologous to the segment [15211, 15430] of SARS-HCoV strain TOR2 from the 13 species of coronaviruses in Fig. 1. 1.The boostrap values are displayed (as percentages) above the node. (B) Phylogenetic tree calculated from the amino acid sequences corresponding to the nucleotide sequences in (A). The boostrap values are displayed (as percentages) above the node. The phylogenetic trees were drawn using the program Treecon for Windows version 1.3.b (Van de Peer and De Wachter, 1994).
Fig. 3
Fig. 3
AluI digestion of amplicons from SARS-HCoV, HCoV-229E and HCoV-OC43. Lanes 1, 3 and 5 (MW): molecular weight markers (Amplisize ladder 50–2000 bp, Bio-Rad Laboratories, Mississauga, Canada); Lane 2: SARS-HCoV (predicted fragments: 126 bp, 94 bp); Lane 4: HCoV-229E (predicted fragments: 101 bp, 86 bp, 33 bp); Lane 6: HCoV-OC43 (predicted fragment: 220 bp).
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