TABLE 1.
Real-time multiplex assays for respiratory virusesa
| Multiplex | Virus assay (reference)b | Primer/probe name | Primer/probe target | Sequence (5′-3′) | Analytical sensitivity (no. of copies per reaction) |
|---|---|---|---|---|---|
| 1 | InfA (22) | Pkz/Glxo FluAfor | Matrix gene | AAGACCAATCCTGTCACCTCTGA | 5 |
| Inf A R/A probe | VIC-TTCACGCTCACCGT-MGB | ||||
| Pkz/Glxo FluA Rev | CAAAGCGTCTACGCTGCAGTCC | ||||
| InfB (22) | GSK FluB for | Matrix gene | GAGACACAATTGCCTACCTGCTT | 1 | |
| GSK FluB Pro MGB | FAM-GCAAAGCAGAACTAGC-MGB | ||||
| GSK FluB Rev4 | TCTTTCCCACCGAACCAACA | ||||
| GSK FluB Rev5 | TCTTTCCCACCAAACCAACA | ||||
| 2 | CoV OC43 | BC OC43 N-gene For | N gene | GTTGTACAGGATGTGGGA | <10 |
| BC OC43 N-gene probe | FAM-CTGGATACCAGGAGTT-MGB | ||||
| BC OC43 N-gene Rev | CGAACTTAGTCGTCATGT | ||||
| CoV 229E | BC 229E N-gene For | N gene | TGCATTTTTATTATCTTGGC | <10 | |
| BC 229E N-gene probe | FAM-CTGTTGATGGTGCTAAA-MGB | ||||
| BC 229E N-gene Rev | TGAATTCTTGCGCCTAAC | ||||
| CoV NL63 (4) | Fouchier et al. (4), set 1 For | N gene | TGTCAACGAGGTTTTGCATTAAAT | <10 | |
| Fouchier et al. (4), set 1 probe | FAM-ATGCGTTTAGCGCATGA-MGB | ||||
| Fouchier et al. (4), set 1 Rev | ACTGGCCTACCATTGTGTGTAAGA | ||||
| CoV HKU1 | HKU1 N gene564 F | N gene | GGCTCAGGAAGGTCTGCTTCTA | <10 | |
| HKU1 N Gene588 Tpr | FAM-AGTCGACCAGGTTCACGTT-MGB | ||||
| HKU1 N Gene 631 R | CGATTATTGGGTCCACGTGAT | ||||
| 3 | RSV group A (21) | RSV van Elden RSA-1 | N gene | AGATCAACTTCTGTCATCCAGCAA | ND |
| RSV van Elden RSA Probe | FAM-CACCATCCAACGGAGCACAGGA GAT-MGB | ||||
| RSV van Elden RSA-2 | TTCTGCACATCATAATTAGGAGTATCAAT | ||||
| RSV group B (21) | RSV van Elden RSB-1 | N gene | AAGATGCAAATCATAAATTCACAGGA | ND | |
| RSV van Elden RSB Probe | FAM-ATATGCTATGTCCAGGTTAG-MGB | ||||
| RSV van Elden RSB-2 | TGATATCCAGCATCTTTAAGTATCTTTATAGTG | ||||
| 4 | PIV types 1 and 3 | HPIV 1+3 For3 | L gene | AGTGCAATMCATCTWGCRGCTGTTA | PIV type 1 = 16, PIV type 3 = 258 |
| HPIV3MQAFor4 | ATTATAAGTAGCAAGATGTTCATATATAGCAAA | ||||
| HPIV 1+3 Probe3 | FAM-CAAGGAGAYAATCAAGCYATA-MGB | ||||
| HPIV3MQAPro4 | FAM-GATGGGAGAATTCTTC-MGB | ||||
| HPIV1+3 Rev7 | GTGATRTCTTCATAGACGTGAGTCTTT | ||||
| HPIV3MQARev8 | ACCARAAGACACATCTAGATAATGCTTT | ||||
| PIV type 2 | HPIV2 MQA For2 | Phosphoprotein gene | CCCAGCATCTGCGGAGAAT | 1,455 | |
| HPIV2 MQA Pro2 | FAM-AAGGAGATCATTGAGCTCT-MGB | ||||
| Para2MQAPro2b | FAM-AGGGAGATCATTGAGCTCT-MGB | ||||
| HPIV2 MQA Rev2 | AAGGCGAAGATCAAGTCCCTTT | ||||
| HPIV2MARev2a | CTACAGTCTGAAGGCGAAGATCAAG | ||||
| Para2MQARev2b | AAGGCGAAGATCAAGCCCCTTT | ||||
| PIV types 4a and 4b | HPIV4MQAPhosFor4 | Phosphoprotein gene | CACTCAACAAATYAAAGGTTCACTTGC | 356 | |
| HPIV4MQAPhosPro4 | FAM-ATTGAGGGCCTAATCAC-MGB | ||||
| HPVI4MQAPhosRev4 | GACTCCAGGGTCCATTATTTTCA | ||||
| 5 | AdV | Ad Hex MQA5 For | Hexon gene | CGCAGTGGKCTTACATGCACATC | Species A = 34, species B = 11, species C = 9, species D = 12, species E = 5 |
| Ad Hex MQA1 probe | Hexon gene | FAM-CCGGGCTGGTGCAGTT-MGB | |||
| Ad Hex MQA2 probe | Hexon gene | FAM-CGGGTCTGGTGCAGTT-MGB | |||
| Ad Hex 31 probe | Hexon gene | FAM-CCGGTCTGGTGCAATT-MGB | |||
| Ad Hex B/E probe | Hexon gene | FAM-GCTTCGGAGTACCT-MGB | |||
| Ad Hex A/C/D probe | Hexon gene | FAM-GCCTCGGAGTACCT-MGB | |||
| Ad Hex MQA6 Rev | Hexon gene | CCACTGTGGGGTTTCTAAATTTGTT | |||
| Ad Hex MQA7 Rev | Hexon gene | ACCGTGGGGTTTCTAAACTTGTT | |||
| Ad Hex 31 Rev | Hexon gene | CCACAGTGGGATTCCTAAACTTGTT | |||
| Ad Pent PE15841 For | Penton gene | AAACGTTCCTGCTCTCACAGATC | |||
| Ad Pent PE15939 RProbe | Penton gene | FAM-CAGGTGCGGCGTCT-MGB | |||
| Ad Pent PE15963 Rev | Penton gene | CCCAGGGCCTTGTARACGTA | |||
| 6 | MPV (11) | Forward | CATATAAGCATGCTATATTAAAAGAGTCTC | MPV-B = 9 | |
| NLN1MGB | FAM-TGCAATGATGAGGGTGTCA-MGB | ||||
| NLN2MGB | FAM-TAATGATGAGGGTGTCACTG-MGB | ||||
| Reverse | CCTATTTCTGCAGCATATTTGTAATCAG | ||||
| 7 | RV (10) | CDC Rhino F | 5′ NCR | Cy+dAGCC+dTGCGTGGC | Clade A = 2-25,c clade B = 13, clade C = 13 |
| CDC Rhino P | FAM-TCCTCCGGCCCCTGAATGYGGC-3BHQ_1 | ||||
| CDC Rhino R | GAAACACGGACACCCAAAGTA | ||||
| 8 | BCR/ABL | BCRABLfor | CCGCTGACCATCAATAAGGAA | ND | |
| BCRABLprobe | NED-AGCCCTTCAGCGGC-MGB | ||||
| BCRABLrev | TGAGGCTCAAAGTCAGATGCTACT |
a
ND, not determined; NCR, noncoding region.
b
The source of the PCR assay is shown by the reference citation. In some instances, the primer and probe sequences listed represent modifications of the published sequences. If no reference citation is provided, the assay was developed for this study. The BCR/ABL primers and probe are from Asuragen, Inc.
c
Two clade A viruses were evaluated. The LOD for rhinovirus type 2, the sequence of which matches the sequences of the primers and probes, was two copies. The LOD for rhinovirus type 33, the sequence of which has one mismatch in the forward primer, was 25 copies.
d
Locked nucleic acid.