Simultaneous detection of chikungunya virus, dengue virus and human pathogenic Leptospira genomes using a multiplex TaqMan® assay

Table 1 List of primers

Name	Fluorochrome	Sequence (5′➔ 3′)	Quencher	Nt	Tm (°C)	Target
CHIK-F		AAGCTYCGCGTCCTTTACCAAG		22	62.1–64.2	E1
CHIK-R		CCAAATTGTCCYGGTCTTCTT		21	57.5–59.5
CHIK-P	FAM	CCAATGTCYTCMGCCTGGACACCTTT	BHQ1	26	67.9–71.1
DF		AGGACTAGAGGTTAGAGGAGA		21	59.5
DRa		CGTTCTGTGCCTGGAATGAT		20	58.4
DRb		CGCTCTGTGCCTGGATTGAT		20	60.5
DP	HEX	CCA + GA + GAT + CCT + GCT	BHQ1	14	43.7 + LNA = 67	3′NC
Lep23S-F		AGAATTGGGATGAGGTGTGGATAG		24	63.6
Lep23S-R		CTACCCCCGCAACTAAACAACTG		23	64.6
Lep23S-P	ATTO647N	CCG + AAA + TAG + GTT + TA + GG + CCT	BHQ3	19	55 + LNA = 72	23S rRNA

Variable melting temperature was indicated for degenerate primers. Y accounts for C/T, M for A/C. Probes are quenched using adequate Black Hole Quenchers (BHQ). Locked Nucleic Acid nucleotides (LNA) are prefixed with a “+” sign and the resulted increase in Tm was indicated following use of Exiqon™ tool for calculation

ISSN: 1471-2180