Taqman allelic discrimination assays for the 5 SNVs were first tested on synthetic templates (Sigma Genosys, Australia) (Table ) and verified using 2 viral isolates, CUHK-W1 [GenBank :AY278554
] and CUHK-Su10 [GenBank :AY282752
]. CUHK-W1 is a SARS-CoV isolate with a G:A:C:T:C motif at the GZ02 reference nucleotide residues 17,564, 21,721, 22,222, 23,823, and 27,827, characteristic of SARS-CoV strains isolated before worldwide dissemination of SARS [8
]. On the other hand, CUHK-Su10 demonstrates a T:G:T:T:T motif which is characteristic of SARS-CoV strains isolated after global spread was evident. As evident from figure , the newly developed allelic discrimination assays were able to differentiate the 2 viral isolates and genotype each SNV correctly (Table ).
Figure 1 Allelic discrimination plot of CUHK-Su10 and CUHK-W1. Allelic discrimination at each of the 5 studied SNVs described in the text as demonstrated using the synthetic templates and cDNA from CUHK-Su10 and CUHK-W1 Vero cell culture isolates is presented (more ...)
Following initial development and optimization, the allelic discrimination assays were used to genotype SARS-CoV in clinical samples. We were able to successfully determine the SARS-CoV genotypes in all 30 samples. Genotypes of virus isolates at the 5 SNV positions are shown in Table . SARS-CoV from all but seven cases showed the T:G:T:T:T motif resembling that of the CUHK-Su 10 isolate. For the remaining seven cases, no allelic signal was detected at SNV position 27,827 (Figure ), leading to a T:G:T:T:/ motif.
Genotype of SARS-CoV culture isolates from 30 patients determined by Taqman Allelic Discrimination assays
The SARS-CoV genotypes isolated from the 30 patients were also confirmed by direct sequencing. The sequencing results were fully concordant with that based on the allelic discrimination assays at all the 5 SNVs. The seven samples which gave no allelic signal by the allelic discrimination assay at SNV 27,827 showed a shortened amplicon encompassing the region. Direct sequencing of this short amplicon revealed a deletion of 386 nt identical to a SARS-CoV deletion variant previously reported by our group [8
]. This deletion variant was first isolated from a discrete cohort of 15 epidemiologically related SARS patients [9
]. In the previous cohort of patients, the origin of the deletion variant was traceable to mid-April 2003 in two patients residing in an estate, T, in Hong Kong with subsequent spread predominantly at the North District Hospital, Hong Kong [9
]. To further determine if the newly identified cases were epidemiologically related to the original patient cohort, the case histories were reviewed.
The seven patients had fever onset between April 4 to 15, 2003 which predated the disease onset dates of all cases in the original patient cohort. All but one patient presented to hospital A initially for other medical conditions without fever or evidence of chest infection and appeared to have acquired SARS nosocomially during admission. The remaining patient was a resident at Estate T and presented to hospital A with symptoms and signs of chest infection on April 8, 2003 and SARS was subsequently confirmed. Thus, it appeared that we have identified another cohort of patients harboring the SARS-CoV variant with the 386-nt deletion. It is interesting to note that this study provided additional anecdotal evidence pointing to Estate T as a propagation site for the deletion variant. In addition, we were able to trace the emergence of this deletion variant to early April 2003, weeks before the first appearance reported previously [9
Our study has clearly demonstrated the feasibility of using allelic discrimination assays as a method for genetic characterization of SARS-CoV genotypes in patients. It is particularly useful when there is already extensive sequence information. Direct sequencing is still the gold standard for identifying new sequence variations when new agents of infectious disease continue to emerge and old ones reemerge. Once the variations have been identified, allelic discrimination assay is more efficient and suitable for large-scale population investigations. A recent study illustrated the use of mass spectrometry-based technology in characterizing SARS sequence variations [14
]. However, this method requires post-PCR manipulations and the availability of specialized equipment. On the other hand, allelic discrimination assays have been widely used in the study of associations between single nucleotide polymorphisms and diseases such as cancers [15
] and rheumatoid arthritis [16
]. The validity of the approach for single nucleotide polymorphism genotyping has been previously demonstrated [17
]. Thus, this study further extended the usefulness of allelic discrimination approach based on fluorogenic oligonucleotide probes. The approach provides a rapid and simple means to accurate genotype screening, making it ideal for epidemiological investigations.