Table 1 Oligonucleotide sequences
Assay | Organism | Gene target | Primer (5’-3’) | Probe (5’-3’) | Product (bp) |
|---|---|---|---|---|---|
Multiplex asymmetric PCR 1 | Acinetobacter baumannii | Citrate synthase(gltA) | F:CTCTGCTGGTATCTCTGCTCT | TGTTGCTGAGTTCATGGAAAAAGTTAAACG-TTTTTTTTTTTT-amino | 222 |
R:Biotin-TGCTTCAAGAACTTCGTCAC | |||||
Staphylococcus aureus | Thermostable nuclease (nuc) | F:AGCGATTGATGGTGATAC | CAAAGAACTGATAAATATGGACGTGGC-TTTTTTTTTTTT-amino | 276 | |
R: Biotin-AAGCCTTGACGAACTAAAG | |||||
Enterococcus faecalis | D-alanine--D-alanine ligase (ddl) | F:GAACGACCACAAAATAAAG | TTACATGGGCCAAATGGTGAAGATGGAACA-TTTTTTTTTTTT-amino | 275 | |
R: Biotin-GCCAACAGTTTGTAAAAGAT | |||||
Mycoplasma pneumoniae | Adhesin protein (P1) | F:TGGTCCTACACCGACTTACA | TGAGGTGAATGGGTTGTTGAATCCG-TTTTTTTTTTTT-amino | 107 | |
R: Biotin-TTCCCAAAATAGGTTTCCAC | |||||
Multiplex asymmetric PCR 2 | Enterobacter cloacae | Molecular chaperone (dnaJ) | F:GTCACCAAAGAGATCCGTA | GCAGGCGATCTGTACGTTCAGG-TTTTTTTTTTTT-amino | 524 |
R: Biotin-CGCATGCGGAACAGCTT | |||||
Burkholderia cepacia | Recombinase A-like (recA) | F:ATATCCAGGTCGTCTCCA | TGGTGCGCTCGGGCTCGATCGACA-TTTTTTTTTTTT-amino | 453 | |
R: Biotin-AGTTCGTGCGCTTGATCGT | |||||
Klebsiella pneumoniae | Malate dehydrogenase (mdh) | F:GCGTGGCGGTAGATCTAAGTCATA | AAAGCCGGCGTGTACGATAA-TTTTTTTTTTTT-amino | 364 | |
R: Biotin-TTCAGCTCCGCCACAAAGGTA | |||||
Escherichia coli | Bacterial alkaline phosphatase (phoA) | F:CCAACGATTCTGGAAATGG | CGCCAAATCCGCAACGTAATGACAGTGTACCAACCC-TTTTTTTTTTTT-amino | 527 | |
R: Biotin-CAATGGCTTTGTCGGTCAT | |||||
Pseudomonas aeruginosa | Exotoxin A (toxA) | F:TCATCCACGAACTGAACG | TTGTGCCTGCTCGACCCGCTGGACGGGGTCTACAACTACCTCGCCCAG-TTTTTTTTTTTT-amino | 325 | |
R: Biotin-ATCTTGCCTTCCCAGGTAT | |||||
Stenotrophomonas maltophilia | Chitinase A (chitA) | F:TCAAGCAGCTCAAGGCCAA | TACCACCCGTACCTGGAC-TTTTTTTTTTTT-amino | 435 | |
R: Biotin-TGGAAGTCGTAGGTCATC | |||||
Multiplex asymmetric PCR 3 | Haemophilus infuenzae | Adhesin protein (P6) | F:TCTAACAACGATGCTGCAGG | GAACGTGGTACACCAGAATACAACATCGC-TTTTTTTTTTTT-amino | 296 |
R: Biotin-CCAGCATCAACACCTTTACC | |||||
Legionella pneumophila | Macrophage infectivity potentiator (mip) | F: CTACAGACAAGGATAAGT | ATAGCATTGGTGCCGATTTGGGGAAGAA-TTTTTTTTTTTT-amino | 108 | |
R: Biotin-CTTGCATGCCTTTAGCCA | |||||
Enterococcus faecium | D-alanine--D-alanine ligase (ddl) | F:GCTAAAGCCACGCCTTCTA | TCCTTTTTCCGTCATCAGTATAAAGTATAG-TTTTTTTTTTTT-amino | 264 | |
R: Biotin-GGTGACGGATGGAAATGTT | |||||
Chlamydia pneumoniae | Major outer membrane protein (ompA) | F:GATCCGCTGCTGCAAACTATACT | ACTGCCGTAGATAGACCTAACCCGGCCTA-TTTTTTTTTTTT-amino | 85 | |
R:Biotin-GTGAACCACTCTGCATCGTGTAA | |||||
Streptococcus pneumoniae | N-acetylmuramoyl-L-alanine amidase (lytA) | F: CCATCTGGCTCTACTGTGAA | CAAAGTAGTACCAAGTGCCATTGATTTTC-TTTTTTTTTTTT-amino | 433 | |
R: Biotin-GAGAACGGCTTGACGATT | |||||
Bacteria (universal primers) | 16S rDNA | F:AGAGTTTGATCMTGGCTCAG(M=A/C) | 575 | ||
R: Biotin-CGTATTACCGCGGCTGCTG | |||||
Acinetobacter spp. | 16S rDNA | CCTAGAGATAGTGGACGTTAC-TTTTTTTTTTTT-amino | |||
Staphylococcus spp. | 16S rDNA | ACATATGTGTAAGTAACTGTGCACATCTTGACGGTA-TTTTTTTTTTTT-amino | |||
Enterococcus faecalis | 16S rDNA | AGTGCTTGCACTCAATTGGAAAGAGGAGTGG-TTTTTTTTTTTT-amino | |||
Mycoplasma spp. | 16S rDNA | GACCTGCAAGGGTTCGT-TTTTTTTTTTTT-amino | |||
Burkholderia spp. | 16S rDNA | TTGGCTCTAATACAGTCGG-TTTTTTTTTTTT-amino | |||
Klebsiella spp./Escherichia spp./Enterobacter spp. | 16S rDNA | GGTTAATAACCTCATCGATTGACGTTACCCTGC-TTTTTTTTTTTT-amino | |||
Pseudomonas spp. | 16S rDNA | TTGCTGTTTTGACGTTAC-TTTTTTTTTTTT-amino | |||
Stenotrophomonas spp. | 16S rDNA | CCAGCTGGTTAATACCCGGTTGGGA-TTTTTTTTTTTT-amino | |||
Haemophilus spp. | 16S rDNA | GAGGAAGGTTGATGTGTTA-TTTTTTTTTTTT-amino | |||
Legionella spp. | 16S rDNA | AGGGTTGATAGGTTAAGAGCTGATTAA-TTTTTTTTTTTT-amino | |||
Enterococcus faecium | 16S rDNA | CAAGGATGAGAGTAACTGTTCATCCC-TTTTTTTTTTTT-amino | |||
Chlamydia spp. | 16S rDNA | CCGAATGTAGTGTAATTAGGC-TTTTTTTTTTTT-amino | |||
Streptococcus spp. | 16S rDNA | TGTGAGAGTGGAAAGTTCACACTG-TTTTTTTTTTTT-amino | |||
Positive control | Bacteria | 16S rDNA | ACTCCTACGGGAGGCAGCAG-TTTTTTTTTTTT-amino | ||
Negetive control 1 | Human | Epidermal growth factor receptor (EGFR) | TCAGAGCCTGTGTTTCTACCAA-TTTTTTTTTTTT-amino | ||
Negetive control 2 | Virus | Neuraminidase (NA) | CATCAATAGGGTCCGATA-TTTTTTTTTTTT-amino | ||
Negetive control 3 | Fungus | Internal transcribed spacer2 (ITS2) | CGAACGCAAATCAATCTTTTTCCAGGT-TTTTTTTTTTTT-amino | ||
Quality controla | 20T | Biotin-TTTTTTTTTTTTTTTTTTTT-amino |