Identification of the severe acute respiratory syndrome coronavirus by simultaneous multigene DNA sequencing

Abstract

The recent severe acute respiratory syndrome (SARS) outbreak resulted in calls for an accurate diagnostic test that can be used not only for routine testing but also for generating nucleotide sequences to monitor the epidemic. Although the identity of the SARS coronavirus (SARS-CoV) genome was confirmed by DNA sequencing, it is impractical to sequence the entire 29-kb SARS-CoV genome on a routine basis. Therefore, alternative assay methods such as the enzyme-linked immunosorbent assay and PCR have been pursued for routine testing, primarily to resolve probable cases. We report here a modification of standard DNA sequencing technology for accurate identification of SARS-CoV in routine testing. Instead of requiring the sequencing of the whole SARS-CoV genome, our modification enables the simultaneous sequencing of three regions of the SARS-CoV genome, the spike protein-encoding gene (35 nucleotides), gene M (43 nucleotides), and gene N (45 nucleotides), in a single electropherogram. Comparing these nucleotide sequences to DNA databank entries (National Institutes of Health) conclusively identified them as SARS-CoV sequences.

FIG. 1.

Stages of the MultiGEN process and the basic scientific principles. These include simultaneous generation of three amplicons from the spike protein-encoding gene, gene M, and gene N and simultaneous sequencing of the 3′ end of each of the amplicons.

FIG. 2.

(A) Agarose gel electrophoresis showing amplicons. Lane 1, spike gene amplicon (158 bp); lane 2, gene M amplicon (190 bp); lane 3, gene N amplicon (176 bp). (B) Electropherogram showing nucleotide sequences from three regions of the SARS-CoV genome: the spike protein-encoding gene (35 nucleotides), gene M (43 nucleotides), and gene N (45 nucleotides).