Table 1.
Primers and probes usedin qPCR assays
| Assay | Gene | Primer and Probe sequences | Position | Amplicon size (bp) | Reference |
|---|---|---|---|---|---|
| Ao |
16S rRNA |
Forward primer 5'- CTACGCATTTCACCGCTACAC -3' |
437-457 |
265 |
[33] |
| Reverse primer 5'- GGGGAAGAACACGGATAGGA -3' |
702-483 |
||||
| TBL |
16S rRNA |
Forward primer 5'- TCCTACGGGAGGCAGCAGT -3' |
331-349 |
466 |
[6,36] |
| Reverse primer 5'- GGACTACCAGGGTATCTAATCCTGTT -3' |
797-772 |
||||
| Hi |
hpd |
Forward primer 5’- GGTTAAATATGCCGATGGTGTTG -3’ |
822-844 |
151 |
[37,38] |
| Reverse primer 5’- TGCATCTTTACGCACGGTGTA -3’ |
972-953 |
||||
| Probe 5’Hex- TTGTGTACACTCCGT “T” GGTAAAAGAACTTGCAC -SpC6 -3’* |
928-896 |
||||
| Spn |
lytA |
Forward primer 5'- TCTTACGCAATCTAGCAGATGAAGC -3' |
306-326 |
101 |
[6] |
| Reverse primer 5'- GTTGTTTGGTTGGTTATTCGTGC -3' |
406-386 |
||||
| Probe 5'- [6-FAM]-TTTGCCGAAAACGCTTGATACAGGG -[TAMRA] -3' |
354-330 |
||||
| Mc | copB | Forward primer 5'- GTGAGTGCCGCTTTTACAACC -3' |
50-70 |
72 | [6] |
| Reverse primer 5'- TGTATCGCCTGCCAAGACAA -3' | 121-102 |
Ao = A. otitidis. TBL = Total bacterial load. Spn = S. pneumoniae. Hi = H. influenzae. Mc = M. catarrhalis. 16S rRNA gene position numbers are based on E. coli numbering. * Inverted commas in the primer sequence of the hpd probe indicate the position of the Black Hole Quencher.