Development of a combined RLEP/16S rRNA (RT) qPCR assay for the detection of viable M. leprae from nasal swab samples

Table 2 Sequences of applied primers and probes

Test	Primer/probe^a	Sequence (5′- 3′)^b	Nucleotide position^c	Amplicon size^d
RLEP PCR^e	RL - F2	ACC TGA TGT TAT CCC TTG CAC	39,741–39,761	167 bp
RLEP PCR^e	RL - R2	CGC TAG AAG GTT GCC GTA TG	39,908–39,889	167 bp
RLEP qPCR	RLEP - F	GCA GTA TCG TGT TAG TGA A	39,839–39,857	69 bp
	RLEP - R	CGC TAG AAG GTT GCC GTA TG	39,908–39,889
	RLEP - P	6FAM- CGC CGA CGG CCG GAT CAT CGA -BBQ	39,885–39,865
16S rRNA RT qPCR	ML16S rRNA TaqF	GCA TGT CTT GTG GTG GAA AGC	1,341,385–1,341,405	70 bp
	ML16S rRNA TaqR	CAC CCC ACC AAC AAG CTG AT	1,341,455–1,341,436
	ML 16S - TP2	6FAM- CCA TCC TGC ACC GCA AAA A -BBQ	1,341,424–1,341,406
GAPDH^f (RT) qPCR	GAPDH fwd	GAA GGT GAA GGT CGG AGT C	194–212	225 bp
	GAPDH rev	GAA GAT GGT GAT GGG ATT TC	419–400
	GAPDH TM	FAM-CAA GCT TCC CGT TCT CAG CCT -BBQ	390–370

^aF Forward primer, R Reverse primer, P/TP/TM Hydrolysis probes (TibMolBiol, Berlin, Germany)
^b Hydrolysis probe with 6-Caboxyfluorescein fluorescent dye (6FAM) and BlackBerry Quencher (BBQ)
^c Nucleotide positions are provided for the first copy of the respective amplicon in Mycobacterium leprae Br4923 (GenBank accession number FM211192.1). For GAPDH qPCR nucleotide positions are provided for the copy in Homo sapiens glyceraldehyde-3-phosphate dehydrogenase (GenBank accession number NM_002046.5) [25]
^d bp = base pairs
^e Direct DNA sequencing was conducted with the forward primer RL-F2. The sequence encompassed the region amplified by RLEP qPCR
^f GAPDH = glyceraldehyde-3-phosphate-dehydrogenase

ISSN: 1471-2334