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. 2013 Jul;51(7):2280–2287. doi: 10.1128/JCM.00335-13

Table 1.

Real-time PCR assay panel design

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)
a

References are given in parentheses.

b

FL, fluorescein; LC640, LightCycler Red 640; LC705, LightCycler Red 705.

c

Calculated based on 354 isolates.

d

tuf, elongation factor Tu.

e

PanStaphHP1 and HP2, probes that detect Staphylococcus aureus and SCN.

f

SAtufHP1 and HP2, S. aureus-specific probes.

g

rpoB encodes the β-subunit of RNA polymerase.

h

S. agalactiae positive, with a Tm of 63°C, compared to Tms of <60°C for other Streptococcus spp.

i

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.

j

The four negative isolates were Morganella morganii, Pantoea agglomerans, Providencia rettgeri, and Serratia liquefaciens.

k

The Actinomyces spp. had Tms of ≤56°C, compared to 61 to 63°C for Propionibacterium spp.

l

Corynebacterium amycolatum positive, with a Tm of 58°C.

m

gyrB encodes DNA gyrase B.

n

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).

o

Polyphosphate kinase gene, corresponding to pvdS2 of C. jeikeium K411 (NC_007164).

p

Corynebacterium group F1 negative.

q

leuB encodes β-isopropylmalate dehydrogenase.

r

B. caccae negative.