Patent ID: 7709188

Claim:
A molecular-beacon-based multi-allelic real-time reverse transcription polymerase chain reaction (RT-PCR) multiplex assay for a Sever Acute Respiratory Syndrome (SARS) virus comprising: (1) obtaining a sample; (2) isolating RNA from the sample; (3) placing the isolated RNA in a tube along with primers specific for spike (S), envelope (E), membrane (M), and nucleocapsid (N) genes of the SARS virus, wherein the S primer includes SEQ ID NO:18, the E primer includes SEQ ID NO:38, the M primer includes SEQ ID NO:59, and the N primer includes SEQ ID NO:80, (4) reverse transcribing the isolated RNA in the presence of a reverse transcriptase to form a cDNA unique for each of the S, E, M and N SARS viral genes, (5) real-time amplifying the SARS cDNA in the presence of four distinct types of molecular beacons, each labeled with the same fluorophore and specific for a different SARS coronavirus (CoV) gene selected from S, E, M and N viral genes, and four S, E, M and N gene-specific primer pairs, wherein the molecular beacons are: for S gene: LK249 5′-FAM-CCCACGCCAGAAGGTAGATCACGAACTACACGTGGG-3′-Dubcyl (SEQ ID NO:14); for E gene: LK253 5′-FAM-CCTCCGCACGAAAGCAAGAAAAAGAAGTACGCCGGAGG-3′-Dubcyl (SEQ ID NO:34); for M gene: LK257 5′-FAM-CCTCCGACCCAATTAATTCTGTAGACAGCAGCCGGAGG-3′-Dubcyl (SEQ ID NO:55); and for N gene: LK261 5′-FAM-CCTCCGTACCATCTGGGGCTGAGCTCTTFCATCGGAGG-3′-Dubcyl (SEQ ID NO:76); wherein the S, E, M and N gene-specific primer pairs are: for S gene LK251 5′-CTCTATGTTTATAAGGGCTATCAACC-3′ (SEQ ID NO:16) LK252 5′-CCAAGAGGCAACTTAAAAATAGGTTTC-3′ (SEQ ID NO:17); for E gene LK255 5′-CGGAAGAAACAGGTACGTTAATAG-3′ (SEQ ID NO:36) LK256 5′-AAGCGCAGTAAGGATGGCTA-3′ (SEQ ID NO:37); for M gene LK259 5′-CTTGTTTTCCTCTGGCTCTTG-3′ (SEQ ID NO:57) LK260 5′-CAAGCCATTGCAATCGCAATC-3′ (SEQ ID NO:58); and for N gene LK263 5′-ACGAGTTCGTGGTGGTGAC-3′ (SEQ ID NO:78) LK264 5′-CGTAGGGAAGTGAAGCTTC-3′ (SEQ ID NO:79); and (6) measuring fluorescence, which is a collective measure of the presence of the SARS virus.