Abstract
BackgroundThe Severe Acute Respiratory Syndrome (SARS) was a newly emerged infectious disease which caused a global epidemic in 2002–2003. Sequence analysis of SARS-coronavirus isolates revealed that specific genotypes predominated at different periods of the epidemic. This information can be used as a footprint for tracing the epidemiology of infections and monitor viral evolution. However, direct sequencing analysis of a large number of clinical samples is cumbersome and time consuming. We present here a simple and rapid assay for the screening of SARS-coronavirus genotypes based on the use of fluorogenic oligonucleotide probes for allelic discrimination.MethodsThirty SARS patients were recruited. Allelic discrimination assays were developed based on the use of fluorogenic oligonucleotide probes (TaqMan). Genotyping of the SARS-coronavirus isolates obtained from these patients were carried out by the allelic discrimination assays and confirmed by direct sequencing.ResultsGenotyping based on the allelic discrimination assays were fully concordant with direct sequencing. All of the 30 SARS-coronavirus genotypes studied were characteristic of genotypes previously documented to be associated with the latter part of the epidemic. Seven of the isolates contained a previously reported major deletion but in patients not epidemiologically related to the previously studied cohort.ConclusionWe have developed a simple and accurate method for the characterization and screening of SARS-coronavirus genotypes. It is a promising tool for the study of epidemiological relationships between documented cases during an outbreak.
Highlights
The Severe Acute Respiratory Syndrome (SARS) was a newly emerged infectious disease which caused a global epidemic in 2002–2003
We demonstrate the feasibility of the adoption of allelic discrimination assays based on the use of fluorogenic oligonucleotide probes for the genotyping of SARS-CoV isolates
Genotype analysis by Taqman allelic discrimination assay We focused on the development of allelic discrimination assays for the five previously described characteristic single nucleotide variations (SNV) [8]
Summary
The Severe Acute Respiratory Syndrome (SARS) was a newly emerged infectious disease which caused a global epidemic in 2002–2003. Sequence analysis of SARS-coronavirus isolates revealed that specific genotypes predominated at different periods of the epidemic. This information can be used as a footprint for tracing the epidemiology of infections and monitor viral evolution. In a large-scale phylogenetic analysis of SARS-CoV sequences [8], a 5-nucleotide motif at the GZ02 [GenBank :AY390556] reference nucleotide residues 17,564, 21,721, 22,222, 23,823, and 27,827 was identified to be most useful for distinguishing the major SARS-CoV genotypes These major viral genotypes predominated at different periods of the epidemic [8]. We demonstrate the feasibility of the adoption of allelic discrimination assays based on the use of fluorogenic oligonucleotide probes for the genotyping of SARS-CoV isolates
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
