Table 1.
Primers and probes used at laboratory 1
| Pathogen identifieda and primer/probe | Primer/probe sequence (5′ → 3′) | Concn (nM) | Comments | Target gene,b position (comment) |
|---|---|---|---|---|
| INF-A | Pan-INF-A PCR | |||
| Primer 1 | AAG ACC AAT CCT GTC ACC TCT GA | 900 | M1/2, 169–191 | |
| Primer 2 | CAA AGC GTC TAC GCT GCA GTC C | 900 | M1/2, 263–242 | |
| Probe | TTT GTG TTC ACG CTC ACC GTG CC | 150 | M1/2, 209–231 | |
| INF-B | ||||
| Primer 1 | AAA TAC GGT GGA TTA AAC AAA AGC AA | 300 | HA, 970–995 | |
| Primer 2 | CCA GCA ATA GCT CCG AAG AAA | 300 | HA, 1119–1139 | |
| Probe | CAC CCA TAT TGG GCA ATT TCC TAT GGC | 100 | HA, 1024–1050 | |
| PIV-1 | ||||
| Primer 1 | TGA TTT AAA CCC GGT AAT TTC TCA T | 900 | HN, 375–399 | |
| Primer 2 | CCT TGT TCC TGC AGC TAT TAC AGA | 900 | HN, 456–433 | |
| Probe | ACG ACA ACA GGA AAT C | 100 | HN, 413–428 | |
| PIV-2 | ||||
| Primer 1 | AGG ACT ATG AAA ACC ATT TAC CTA AGT GA | 300 | F and HN, 2852–2880 | |
| Primer 2 | AAG CAA GTC TCA GTT CAG CTA GAT CA | 900 | F and HN, 3006–2983 | |
| Probe | ATC AAT CGC AAA AGC TGT TCA GTC ACT GCT ATA C | 75 | F and HN, 2885–2918 | |
| PIV-3 | ||||
| Primer 1 | TGA TGA AAG ATC AGA TTA TGC AT | 900 | HN, 840–862 | |
| Primer 2 | CCG GGA CAC CCA GTT GTG | 300 | HN, 1088–1071 | |
| Probe | TGG ACC AGG GAT ATA CTA CAA AGG CAA AAT AAT ATT TCT C | 75 | HN, 984–1023 | |
| RSV | RSV A+B PCRc | |||
| Primer A1 | AGA TCA ACT TCT GTC ATC CAG CAA | 900 | N, 53–76 (serotype A) | |
| Primer A2 | TTC TGC ACA TCA TAA TTA GGA GTA TCA AT | 900 | N, 136–108 (serotype A) | |
| Primer B1 | AAG ATG CAA ATC ATA AAT TCA CAG GA | 300 | N, 164–189 (serotype B) | |
| Primer B2 | TGA TAT CCA GCA TCT TTA AGT ATC TTT ATA GTG | 300 | N, 266–234 (serotype B) | |
| Probe A | CAC CAT CCA ACG GAG CAC AGG AGA T | 58.3 | N, 80–104 (serotype A) | |
| Probe B | TTC CCT TCC TAA CCT GGA CAT AGC ATA TAA CAT ACC T | 66.7 | N, 231–195 (serotype B) | |
| HRV | Pan-Rhino PCR | |||
| Primer 1 | GCC TGC GTG GCT GCC | 300 | Polyprotein, 88–102 (A strain) | |
| Primer 2 | CCT GCG TGG CGG CC | 300 | Polyprotein, 122–135 (B strain) | |
| Primer 3 | ACG GAC ACC CAA AGT AGT TGG T | 300 | Polyprotein, 286–265 (A strain) | |
| Primer 4 | ACG GAC ACC CAA AGT AGT CGG T | 300 | Polyprotein, 318–297 (B strain) | |
| Probe A | TCC GGC CCC TGA ATG TGG CTA A | 100 | Polyprotein, 175–196 (A strain) | |
| Probe B | TCC GGC CCC TGA ATG CGG CTA A | 100 | Polyprotein, 207–228 (B strain) | |
| HCOV | 229E, NL63, OC43 | |||
| Primer 1 | CAG TCA AAT GGG CTG ATG CA | 300 | N, 154–173 (229E) | |
| Primer 2 | CAA AGG GCT ATA AAG AGA ATA AGG TAT TCT | 300 | N, 231–201 (229E) | |
| Primer 3 | GCG TGT TCC TAC CAG AGA GGA | 50 | N, 157–177 (NL63) | |
| Primer 4 | GCT GTG GAA AAC CTT TGG CA | 300 | N, 275–256 (NL63) | |
| Primer 5 | CGA TGA GGC TAT TCC GAC TAG GT | 900 | N, 577–599 (OC43) | |
| Primer 6 | CCT TCC TGA GCC TTC AAT ATA GTA ACC | 900 | N, 652–626 (OC43) | |
| Probe A | CCC TGA CGA CCA CGT TGT GGT TCA | 100 | N, 199–176 (229E) | |
| Probe B | ATG TTA TTC AGT GCT TTG GTC CTC GTG AT | 100 | N, 180–208 (NL63) | |
| Probe C | TCC GCC TGG CAC GGT ACT CCC T | 125 | N, 601–622 (OC43) | |
| HADV | Pan-Adeno PCR | |||
| Primer 1 | TTT GAG GTG GAY CCM ATG GA | 225 | Hexon, xd | |
| Primer 2 | TTT GAG GTY GAY CCC ATG GA | 225 | Hexon, yd | |
| Primer 4 | AGA ASG GSG TRC GCA GGT A | 225 | Hexon, x + 105d | |
| Primer 5 | AGA ASG GTG TRC GCA GAT A | 225 | Hexon, y + 105d | |
| Probe A | ACC ACG TCG AAA ACT TCG AA | 100 | Hexon, x + 45d | |
| Probe B | ACC ACG TCG AAA ACT TCA AA | 100 | Hexon, y + 45d | |
| Probe C | ACA CCG CGG CGT CA | 100 | Hexon, x/y + 80d | |
| M. pneumoniae | ||||
| Primer 1 | GGT CAA TCT GGC GTG GAT CT | 50 | P1, 3967–3986 | |
| Primer 2 | TGG TAA CTG CCC CAC AAG C | 300 | P1, 4032–4014 | |
| Probe | TCC CCC GTT GAA AAA GTG AGT GGG T | 125 | P1, 3988–4012 | |
| L. pneumophila | ||||
| Primer 1 | GCA ATG TCA ACA GCA ATG GC | 300 | MIP, 13–32 | |
| Primer 2 | CGG CAC CAA TGC TAT AAG ACA A | 300 | MIP, 94–73 | |
| Probe | CAA CCG ATG CCA CAT CAT TAG CTA CAG ACA | 100 | MIP, 35–64 | |
| C. pneumoniae | ||||
| Primer 1 | AAA CAA TTT GCA TGA AGT CTG AGA A | 900 | MOMP, 756–732 | |
| Primer 2 | TCC GCA TTG CTC AGC C | 300 | MOMP, 631–646 | |
| Probe | TAA ACT TAA CTG CAT GGA ACC CTT CTT TAC TAG G | 75 | MOMP, 667–700 |
a
INF, influenza virus; PIV, parainfluenza virus; RSV, respiratory syncytial virus; HRV, human rhinovirus; HCOV, human coronavirus; HADV, human adenovirus.
b
M1/2, matrix 1 and 2; HA, hemagglutinin; HN, hemagglutinin-neuraminidase; F, fusion; N, nucleoprotein; P1, cytadhesin; MIP, macrophage infectivity potentiator; MOMP, major outer membrane protein.
c
See reference 18a.
d
Start position differs with serotype.