A multiplex one-tube nested real time RT-PCR assay for simultaneous detection of respiratory syncytial virus, human rhinovirus and human metapneumovirus

Table 4 Primers and probes used in this work

Virus	Oligo	Sequence (5’to 3’)	Primer lengths(bp)	Product size(bp)	Gene	References
RSV	F^a	CA + CW + GAA + GA + TG + CWAAT+CATAAATTCA	26	374	N	[8, 26]
	R^a	CW + GA + TC + TRT + CT + CCT + GCTGCTA	21
	F	CACWGAAGATGCWAATCATAAATTCA	26	89
	R	GTATYTTTATRGTGTCTTCYCTTCCTAACC	30
	Probe	FAM-TAATAGGTATGTTATATGCKATGTC-BHQ1	25
HRV	F^a	HC + AA+GYA + CTTCT+GTYWCCCCSG	22	397	5′UTR	[9, 27]
	R^a	GA + AA+CAC + GGA + CA + CCCAAAGTAGT	23
	F	TGGACAGGGTGTGAAGAGC	19	144
	R	CAAAGTAGTCGGTCCCATCC	20
	Probe	Cy5-TCCTCCGGCCCCTGAATG-BHQ3	18
HMPV	F^a	CATATAAG+CA + T + G + C + TA + TATTAAAA+GAGTCTC	30	475	N	[10, 14]
	R^a	GT + GAATATTAA+G + G + CA + C + CTACACATAATAARA	31
	F	CATATAAGCATGCTATATTAAAAGAGTCTC	30	163
	R	CCTATTTCTGCAGCATATTTGTAATCAG	28
	Probe	VIC-TGYAATGATGAGGGTGTCACTGCGGTTG-BHQ1	28

^aA ‘+’ symbol in front of nucleotides indicates the LNA modification; FAM, 6-carboxyfluorescein; Cy5, Cyanine5; VIC, 2’-chloro-7’-phenyl-1,4-dichloro-6-carboxyfluorescein; BHQ, Black Hole Quencher

ISSN: 1743-422X