TABLE 1.

Real-time polymerase chain reaction 5′ exonuclease assays used for pleural fluid specimen testing

Organism	Target gene	Primer (5′ – 3′)		Probe	Ref
		Forward	Reverse
Streptococcus pneumoniae	lytA	ACGCAATCTAGCAGATGAAGCA	TCGTGCGTTTTAATTCCAGCT	AACGCTTGATACAGGGAG*	21
Streptococcus pyogenes	spy	GCACTCGCTACTATTTCTTACCTCAA	GTCACAATGTCTTGGAAACCAGTAAT	CCGCAACTCATCAAGGATTTCTGTTACCA†	23
Haemophilus influenzae	hpd	GGTTAAATATGCCGATGGTGTTG	TGCATCTTTACGCACGGTGTA	TTGTGTACACTCCGTTGGT*	21
Staphylococcus aureus	nuc	AAATTACATAAAGAACCTGCGACA	GAATGTCATTGGTTGACCTTTGTA	AATTTAACCGTATCACCATCAATCGCTTT†	22
Streptococcus intermedius/Streptococcus constellatus	16S rRNA	TGCAAGTAGAACGCACAGGATG	TGCAGTAAATGTTCTTATGCGGTATTAG	CGCGTAGGTAACCTGCCT‡	24
Bacillus atrophaeus (positive control)	atpD	TTGTCTGTGAATCGGATCTTTCTC	CACTTCATTTAGGCGACGATACT	TCCCAATGTTACATTACC*	20

All assays were labelled with fluorescein amidite.

^*Probe modified from published with use of minor groove binder (MGB);

^†ZEN internal quencher (Integrated DNA Technologies, USA) used;

^‡MGB probe used in place of dual fluorescence resonance energy transfer probes, forward and reverse primers lengthened at the 3′ end to increase melting temperatures. Ref Reference; rRNA Ribosomal RNA