Real-Time RT-PCR for the Detection of Lyssavirus Species

Table 2

Primers and probes.

Method	Name	Sequence	Length	Position^a	Product	Reference
hnRT-PCR	JW12-F	ATGTAACACCYCTACAATG	19	55–73		Panning et al., 2010 [22]
	JW6AS1-R1	CAATTGGCACACATTTTGTG	20	660–641	606 bp
	JW6AS2-R1	CAGTTAGCGCACATCTTATG	20	660–641	606 bp
	JW10AS1-R2	GTCATCAATGTGTGATGTTC	20	636–617	582 bp
	JW10AS2-R2	GTCATTAGAGTATGGTGCTC	20	636–617	582 bp
Cloning RT-PCR	TWclon-F	ACGCTTAACRACMAAACCAG	20	1–20		This work
	TWclon-R	TGKATGAARTAAGAGTGWGGRAC	23	933–911	933 bp
Control^1	GAPDH-F	GGCAAGTTCCATGGCACAGT	20	58–72^b		Ravazzolo et al., 2006 [37]
	GAPDH-R	ACGTACTCAGCACCAGCATCAC	22	161–182^b	125 bp
Real-time RT-PCR	RABVD1-F	ATGTAACACCYCTACAATG	19	55–73		Nadin-Davis et al., 2009 [8]
	RABVD1-R	GCMGGRTAYTTRTAYTCATA	20	165–146	111 bp	Nadin-Davis et al., 2009 [8]
	RABVD1-P	5′-FAM-CCGAYAAGATTGTATTYAARGTCAAKAATCAGGT-BHQ1-3′	34	78–111		Nadin-Davis et al., 2009 [8]
	LysGT5-P	5′-YY-AACARGGTTGTTTTYAAGGTCCATAA-BHQ1-3′	26	80–105		Wakeley et al., 2005 [25]
	LysGT6-P	5′-Cy5-ACARAATTGTCTTCAARGTCCATAATCAG-BHQ2-3′	29	81–109		Wakeley et al., 2005 [25]
	ivvWCBV-P	5′-FAM-TCGGATATCACTTCGGGTTTGAGAGTCA-BHQ1-3′	28	141–114		This work
	ivvMok-P	5′-YY-TTGTGTTCAAGGTGAAYAAYCAAGT-BHQ1-3′	25	87–111		This work
	ivvABLV2n-P	5′Cy5-ATTGTCTTTAAGGTCAACAATCAGTT-BHQ2-3′	26	86–111		This work
	ivvLBV-P	5′-FAM-ATTGTTTTCAAAGTYCAYAATCAGGTMGTGTC-BHQ1-3′	32	86–117		This work
	ivvKhujand-P	5′-YY-ACAGAATTGTCTTYAAAGTYMAKAATCA-BHQ1-3′	28	81–108		This work
	ivvSBLV2-P	5′-Cy5-TCWGAGATTATRTCTGGCTTCAAAGACAC-BHQ2-3′	29	141–113		This work
Control^1	Sendai-F	GTCATGGATGGGCAGGAGTC	20	8553–8572^b		Kaiser, 2001 [38]
	Sendai-R	CGTTGAAGAGCCTTACCCAGA	21	8788–8768^b	236 bp
	Sendai-P	5′-FAM-CAAAATTAGGAACGGAGGATTGTCCCCTC-Tamra-3′	29	8720–8748^b

Changed bases referring to the reference are underlined. Wobbled positions are as follows: Y = C/T, X/N = G/A/T/C, W = A/T, S = C/G, R = A/G, M = A/C, K = G/T, H = A/C/T, and B = C/G/T.
^aRabies primer and probe positions are given according to the Pasteur virus genome (accession number X03673).
^bPositions of the GAPDH-primers refer to the GenBank sequence AJ431207; positions of the Sendai-primers refer to the GenBank sequence M30202.
¹Controls for classical and real-time RT-PCR.
FAM: 6-carboxyfluorescein reporter dye, BHQ-1: Black Hole Quencher-1, YY: Yakima Yellow, Cy5: Cyanine 5, BHQ-2: Black Hole Quencher-2, and Tamra: carboxy-tetramethyl-rhodamine.