Validation and utilization of an internally controlled multiplex Real-time RT-PCR assay for simultaneous detection of enteroviruses and enterovirus A71 associated with hand foot and mouth disease

Table 4 Primer and probe sequences and concentration used in single reaction

Name	Sequence (5’→3’)	Final concentration	Note
EAV-F primer	CATCTCTTGCTTTGCTCCTTAG	400 nM	Internal control [15]
EAV-R primer	AGCCGCACCTTCACATTG	400 nM
EAV-probe	FAM-CGCTGTCAGAACAACATTATTGCCCAC-BHQ1	100 nM
ENT-F	CCCTGAATGCGGCTAAT	400 nM	Enterovirus specific primers and probe [14]
ENT-R	ATTGTCACCATAAGCAGCC	400 nM
ENTr-probe	Cy5-ACCCAAAGTAGTCGGTTCCG -BHQ3	200 nM
EV-A71-634 F	GGAGAACACAARCARGAGAAAGA	400 nM	Enterovirus 71 specific primers and probe [8]
EV-A71-743R	ACYAAAGGGTACTTGGAYTTVGA	400 nM
EV-A71-probe	Cyan500-TGATGGGCACDTTCTCRGTGCG-BHQ1	40 nM

Note: FAM = Carboxyfluorescein; Cy5 = Cyanine 5; Cyan500 = Cyan 500 NHS ester; BHQ = black hole quencher; R = A and G; Y = T and C; V = A, C, and G; D = A, G, and T

ISSN: 1743-422X