Table 2.
Oligonucleotide sequences
| Assay | Organism | Gene target | Oligonucleotide | Final reaction concentration (μM) | Reference |
|---|---|---|---|---|---|
| mRT-PCR 1 | Streptococcus pneumoniae | Autolysin (lytA) | Forward: ACGCAATCTAGCAGATGAAGCA | 0.10 | [17] |
| Reverse: TCGTGCGTTTTAATTCCAGCT | 0.10 | ||||
| Probe: YY-TGCCGAAAACGCTTGATACAGGGAG-BHQ1 | 0.05 | ||||
| Haemophilus influenzae | l-Fuculokinase (fucK) | Forward: ATGGCGGGAACATCAATGA | 0.15 | [20] | |
| Reverse: ACGCATAGGAGGGAAATGGTT | 0.15 | ||||
| Probe: FAM-CGGTAATTGGGATCCAT-MGB | 0.10 | ||||
| Staphylococcus aureus | Thermostable nuclease (nuc) | Forward: AGCATCCTAAAAAAGGTGTAGAGA | 0.15 | [22] | |
| Reverse: CTTCAATTTTMTTTGCATTTTCTACCA | 0.15 | ||||
| Probe: TEX-TTTTCGTAAATGCACTTGCTTCAGGACCA-BHQ2 | 0.10 | ||||
| Moraxella catarrhalisa | Outer membrane protein (copB) | Forward: CGTGTTGACCGTTTTGACTTT | 0.15 | Modified from [23] | |
| Reverse: CATAGATTAGGTTACCGCTGACG | 0.15 | ||||
| Probe: Cy5-ACCGACATCAACCCAAGCTTTGG–BHQ3a | 0.10 | ||||
| mRT-PCR 2 | Escherichia colib | Conserved protein, function unknown (yccT) | Forward: ATCGTGACCACCTTGATT | 0.25 | Modified from [24] |
| Reverse: TACCAGAAGATCGACATC | 0.25 | ||||
| Probe: TEX-CATTATGTTTGCCGGTATCCGTTT-BHQ2 | 0.10 | ||||
| Klebsiella pneumoniae | Citrate synthase (gltA) | Forward: AGGCCGAATATGACGAAT | 0.25 | Modified from [24] Probe: this publication |
|
| Reverse: GGTGATCTGCTCATGAA | 0.25 | ||||
| Probe: YY-ACTACCGTCACCCGCCACA-BHQ1 | 0.10 | ||||
| Pseudomonas aeruginosa | DNA gyrase subunit B (gyrB) | Forward: CCTGACCATCCGTCGCCACAAC | 0.25 | [25] | |
| Reverse: CGCAGCAGGATGCCGACGCC | 0.25 | ||||
| Probe: FAM-CCGTGGTGGTAGACCTGTTCCCAGACC-BHQ1 | 0.10 | ||||
| Acinetobacter baumannii | OXA-51-like β-lactamase (blaOXA-51-like) | Forward: TTTAGCTCGTCGTATTGGACT | 0.125 | Modified from [28] | |
| Reverse: CCTCTTGCTGAGGAGTAATTTT | 0.125 | ||||
| Probe: Cy5-TGGCAATGCAGATATCGGTACCCA-BHQ3a | 0.05 | ||||
| Specimen quality control | Human | Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) | Forward: TTGTCTCACTTGTTCTCT | 0.30 | This publication |
| Reverse: ATGGGAGTTGTTTTCTTG | 0.30 | ||||
| Probe: FAM-CTCGTCTTCTGTCATCTCTGCTG-BHQ1 | 0.20 | ||||
| Internal control for inhibition | PhHV | Glycoprotein B (gB) | Forward: GGGCGAATCACAGATTGAATC | 0.30 | [15] |
| Reverse: GCGGTTCCAAACGTACCAA | 0.30 | ||||
| Probe:Cy5-TTTTTATGTGTCCGCCACCATCTGGATC–BHQ3a | 0.10 |
Fluorophores: BHQ, Black Hole Quencher; TEX, Texas Red; YY, Yakima Yellow.
mRT-PCR, multiplex real-time PCR; PhHV, phocine herpes virus.
a
Moraxella catarrhalis assay detects Moraxella lacunata and Moraxella nonliquefaciens.
b
Escherichia coli assay detects Shigella species.