Detection of Eight Respiratory Bacterial Pathogens Based on Multiplex Real-Time PCR with Fluorescence Melting Curve Analysis

Table 1

Primers designed for eight bacteria pathogens used for single and multiplex real-time PCR.

Primer	Gene	Sequence (5′-3′)	Amplicon size (bp)	Calculated Tm^a	Intra-assay Tm^b	Inter-assay Tm^c
A. baumannii-F	iucD	GGCTGGACATCATCAACTGC	193	79.46	78.89 ± 0.03	79.44 ± 0.25
A. baumannii-R		GTCGGCCTGATCTCGTATGA
E. coli-F	uidA	CGACTGGGCAGATGAACATG	225	84.49	84.48 ± 0.09	85.08 ± 0.33
E. coli-R		TACTCCACATCACCACGCTT
K. pneumoniae-F	yfkN	TACACAATCGCCCGTTGAAC	223	87.79	87.23 ± 0.47	88.02 ± 0.68
K. pneumoniae-R		CCCGGTTAGATCCATGGTGA
H. influenzae-F	atoE	CTGGTGTTGCGGCTAAAAGT	168	82.29	82.73 ± 0.21	83.21 ± 0.55
H. influenzae-R		TCATTAACTGGGGCTTCGGT
S. pneumoniae-F	lytA	GCACACTCAACTGGGAATCC	110	80.50	80.10 ± 0.13	80.49 ± 0.41
S. pneumoniae-R		ATGCAACCGTTCCCAACAAT
S. aureus-F	cap5F	AGTCACGTCTCGATCGAACA	175	76.40	76.33 ± 0.25	77.02 ± 0.16
S. aureus-R		GAAACTTGACCACGATCCGG
P. aeruginosa-F	hpmA	AGAAGACCGTAAGCCAGACC	244	91.01	90.92 ± 0.05	90.94 ± 0.14
P. aeruginosa-R		CTACTGGCACCCACTCCTG
M. tuberculosis-F	IS6110	GTCTACTTGGTGTTGGCTGC	108	89	89.52 ± 0.06	89.65 ± 0.03
M. tuberculosis-R		TCAGCTCAGCGGATTCTTCG

^aObtained by using Primer Premier 5.0. ^b,cObtained by EvaGreen MAC of multiplex real-time PCR, mean ± SD, corresponding to nine replicates.