Improved detection of genus-specific Alphavirus using a generic TaqMan® assay

BMC Microbiology

Table 2 List of primers and probes

Name	Fluorochrome	Sequence (5’➔ 3′)	Quencher	Nt	Tm (°C)	Degeneracy
F1		GGTGCGATGATGAAGTCTGG		20	60.5	0
R1		CTATGATATTGACTTCCATGTTCA		24	58.3	0
F2A		ATGATGAARTCIGGIATGTTYYT		23	53.9–59.2	8
F2B		ATGATGAARTCNGGNATGTT		20	50.2–56.4	32
R2A		ATYTTIACTTCCATGTTCATCCA		23	55.5–57.6	2
R3A		ATYTTIACTTCCATRTTCARCCA		23	53.9–59.2	8
R4A		ATYTTIACTTCCATGTTGACCCA		23	57.6–59.2	2
R2B		ATYTTNACTTCCATGTTCATCCA		23	55.5–59.2	8
R3B		ATYTTNACTTCCATRTTCARCCA		23	53.9–60.9	32
R4B		ATYTTNACTTCCATGTTGACCCA		23	57.6–60.9	8
P1	ATTO425	AT + GTT + GTC + GT + CN + CC	BHQ1/LNA™	14	40.8–43.7/66–69	4
P2	ATTO425	AT + GTT + GTC + GT + CIC + CIAT	BHQ1/LNA™	17	47.5/67	0

Variable melting temperature was indicated for degenerate primers. Degenerate nucleotides were shown in bold. Y accounted for C/T, R for A/G, N for A or T or C or G. I was used as an alternative for N. ATTO425 labeled probes were quenched using Black Hole Quencher 1 (BHQ1). Locked Nucleic Acid nucleotides (LNA™) were prefixed with a “+” sign and the resulted increase in Tm was indicated following use of Exiqon™ tool for calculation

ISSN: 1471-2180