A Quantitative Real-Time RT-PCR Assay for the Detection of Venezuelan equine encephalitis virus Utilizing a Universal Alphavirus Control RNA

Table 1

Primers and probes selected for equine encephalitis virus-specific quantitative reverse transcription polymerase chain reaction.

Target	Primer or probe	Sequence ()	Genome position	Reference
Eastern equine encephalomyelitis virus (EEEV)	EEE9391	ACACCGCACCCTGATTTTACA	9391–9411 (s)
	EEE9459c	CTTCCAAGTGACCTGGTCGTC	9459–9439 (as)	[10]
	EEE.9414probe	FAM-TGCACCCGGACCATCCGACCT-TAMRA	9414–9434 (s)
Western equine encephalomyelitis virus (WEEV)	WEE10,248	CTGAAAGTCGGCCTGCGTAT	10,248–10,267 (s)
	WEE 10,314c	CGCCATTGACGAACGTATCC	10,314–10,295 (as)	[10]
	WEE 10,271probe	FAM-ATACGGCAATACCACCGCGCACC-TAMRA	10,271–10,293 (s)
Venezuelan equine encephalomyelitis virus (VEEV) and synthetic calibrator	AlphaVIR966F	TCCATGCTAATGCYAGAGCGTTTTCGCA	151–178 (s)	Modified [14]
	AlphaVIR966R	TGGCGCACTTCCAATGTCHAGGAT	248–225 (as)
	INEID-VEEV probe	FAM-TGATCGARACGGAGGTRGAMCCATCC-TAMRA	193–218 (s)	This study
	VEEV-Coprobe	VIC-CTCCGTTCAATAC-MGB-	180–192 (s)	This study

The synthetic calibrator RNA is specifically detected by the VEEV-Coprobe in combination with the AlphaVIR966F and AlphaVIR966R primers. Y, H, R, and M are designed for degenerative bases, where Y = C/T, H = A/C/T, R = A/G, and M = A/C. Modifications compared to the original sequence as well as novel sequences were indicated in italic font. MGB: minor groove binder; NFQ: Nonfluorescent quencher.