Table 1.
Target organisma | Target gene | Fragment size (bp) | Forward/reverse primer sequences (5′–3′) | Probe sequences (5′–3′)b | Mg2+ concn (mM) | Primer concns (forward/reverse, in μM) | Cycling parameters |
Limit of detection (CFU/ml of sonicate fluid) | % inclusivityc | % cross-reactivityc | ||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Anneal temp (°C) | Anneal time (s) | No. of cycles | ||||||||||
Staphylococcus (22) | tufd | 447 | CAATGCCACAAACTCG/GCTTCAGCGTAGTCTA | ACGGCCTGTAGCAACAGTAC-FL,e LC640-CGACCAGTGATTGAGAATACGTCCe, GGCGATGCTCAATACGAAGAAAAAATC-FL,f LC705-AGAATTAATGGAAGCTGTAGATACf | 3 | 1/1 | 55 | 8 | 36 | 550 SCN IDRL-7,371, 325 S. aureus ATCC 43300 | 100 (201/201) | 0 (0/153) |
Streptococcus | rpoBg | 137 | CCGGHCGTCACGGWAA/CCATACCAAGRTGAAGYTCCATA | GGAACGCCAGTTGATATCATGYTKAAYCC-FL, LC640-CTTGGGGTGCCATCACGKATGAA | 4 | 0.4/4 | 57 | 15 | 35 | 110 S. agalactiae, 115 S. pneumoniae | 100 (15/15)h | 0 (0/339) |
Enterococcus/Granulicatella/Abiotrophia | rpoB | 143 | CGYGAAGCYGGCGATGAAT/AWGGCATRTCTTCTTCYGGC | CAYGAAGGRGATAARATGGCSGG-FL, LC640-GTCAYGGWAATAAAGGGGTYGTWTC | 4 | 0.4/4 | 55 | 8 | 35 | 200 E. faecalis | 100 (14/14)i | 0 (0/340) |
Enterobacteriaceae other than Proteus | rpoB | 185 | TCTGCWATYGAAGAAGGCAACT/ATCAGGGAHGCACCRAC | GCTTGTTCAGCCGCGACC-FL, LC640-GTTGACTACATGGACGTATCCACCCAGC | 3.5 | 0.4/2 | 55 | 8 | 35 | 190 E. coli | 83.3 (20/24)j | 0 (0/330) |
Gram-positive anaerobic cocci | 16S rRNA | 162 | TCGCGTCYSATTAGCTAGT/GCTGCATCAGRGTTYCC | CACATTGGRACTGAGAHACGGY-FL, LC640-ARACTCCTACGGGAGGCAGCAG | 2.5 | 0.4/2 | 57 | 8 | 35 | 10 F. magna | 100 (12/12) | 0 (0/342) |
Propionibacterium/Actinomyces | 16S rRNA | 243 | CGGATTTATTGGGCGTAAAGR/AGGGTATCTAAGCCTGTTCG | GGCGAAGGCGGTTCTCTG-FL, LC640-CCTTTCCTGACGCTGAGRAGCG | 2.5 | 0.4/2 | 55 | 8 | 35 | 200 P. acnes | 100 (52/52)k | 0.3 (1/302)l |
P. aeruginosa (21) | gyrBm | 222 | CCTGACCATCCGTCGCCACAAC/CGCAGCAGGATGCCGACGCC | GGCGAGACCGATGGCT-FL, LC640-GGCACCGAAGTTCACTTCAAGCCG | 2.5 | 0.4/1 | 55 | 8 | 35 | 200 | 100 (10/10) | 0 (0/344) |
Corynebacteriumn (20) | Kinasen,o | 143 (forward/reverse 1), 149 (forward/reverse2) | CGRTTGTACCARGARCGGT/GCACCTSAAYCCSCGT (reverse 1), CAACGAGCACCTSAACCC (reverse 2) | TAGCGCTGGAAGTACCASGAGGT-FL, LC640-GACTCRCGCGGCGACGG, LC705-GACTCGCGCTCGGAAGG | 4 | 0.4/2 | 57 | 10 | 35 | 200 C. tuberculo-stearicum, 440 C. jeikeium | 80 (4/5)p | 0 (0/349) |
Proteus | rpoB | 183 | CTGTCWGCAATTGAAGAAGGTAACT/GGATCAGTGAWGCACCGA | CTTGCCGTCATARAGGYG-FL, LC705-TCAAGYTTATTTAGTCGTGAT | 2.5 | 0.4/2 | 55 | 8 | 35 | 52 P. vulgaris | 100 (4/4) | 0 (0/350) |
B. fragilis group | leuBq | 185 | CGTCCTGAGATAGAACG/GCAGCATTATCCACAAACATATAA | CTTGCTTCCAGTCGTCTATGGA-FL, LC640-CAGATTGCACAAGAAATGGCGCCGC | 2.5 | 0.4/2 | 55 | 8 | 35 | 150 B. fragilis | 92.3 (12/13)r | 0 (0/341) |
References are given in parentheses.
FL, fluorescein; LC640, LightCycler Red 640; LC705, LightCycler Red 705.
Calculated based on 354 isolates.
tuf, elongation factor Tu.
PanStaphHP1 and HP2, probes that detect Staphylococcus aureus and SCN.
SAtufHP1 and HP2, S. aureus-specific probes.
rpoB encodes the β-subunit of RNA polymerase.
S. agalactiae positive, with a Tm of 63°C, compared to Tms of <60°C for other Streptococcus spp.
The G. adiacens (n = 3) and Abiotrophia species isolates had Tms of 58°C and 50°C, respectively, compared to 55°C for the Enterococcus sp. isolate.
The four negative isolates were Morganella morganii, Pantoea agglomerans, Providencia rettgeri, and Serratia liquefaciens.
The Actinomyces spp. had Tms of ≤56°C, compared to 61 to 63°C for Propionibacterium spp.
Corynebacterium amycolatum positive, with a Tm of 58°C.
gyrB encodes DNA gyrase B.
This assay uses one forward and two reverse primers and three probes in the same reaction, primer reverse 1 and probe LC705-GACTCGCGCTCGGAAGG, specific for the resistant species (C. jeikeium and C. urealyticum).
Polyphosphate kinase gene, corresponding to pvdS2 of C. jeikeium K411 (NC_007164).
Corynebacterium group F1 negative.
leuB encodes β-isopropylmalate dehydrogenase.
B. caccae negative.