Table 2.

Sequences of primers and TaqMan probes for WR-QNRT-PCR assays.

Objective	Target	Type	Sequence	PCR product size
First step PCR	Wild M.Tb DNA (MPT64) or W-plasmid	WFl: Outer wild forward primer WRl: Outer wild reverse primer	5′-ATCCGCTGCCAGTCGTCTTCC-3′ Total 21 nucleotides, A:2, T:6, G:4, C:9 (GC% 62) 5′-CTCGCGAGTCT AGGCCAGCAT-3′ Total 21 nucleotides, A:4, T:4, G:6, C:7 (GC% 62)	239 bp
	New internal control (NM-plasmid)	MFl: Outer mutation forward primer MRl: Outer mutation reverse primer	5′-TCGATTCTGTCCCACCGCCGT-3′ Total 21 nucleotides, A:2, T:6, G:4, C:9 (GC% 62) 5′-AGACTCGACGCGTAGTCCTCG-3′ Total 21 nucleotides, A:4, T:4, G:6, C:7 (GC% 62)
	Wild M.Tb DNA (MPT64) or W-plasmid	TqMn-WF2: TaqMan inner wild forward primer TqMn-WR2: TaqMan inner wild reverse primer	5′-GTGAACTGAGCAAGCAGACCG-3′ Total 21 nucleotides, A:7, T:2, G:7, C:5 (GC% 57) 5′-GTTCTGATAATTCACCGGGTCC-3′ Total 22 nucleotides, A:4, T:7, G:5, C:6 (GC% 50)	77 bp
Second step PCR	New internal control (NM-plasmid)	TqMn-MF2: TaqMan inner mutation forward primer TqMn-MR2: TaqMan inner mutation reverse primer	5′-AGATCGGATAGCCAGCACGGA-3′ Total 21 nucleotides, A:7, T:2, G:7, C:5 (GC% 57) 5′-TGCGCTGCGTCGACATATTCT A-3′ Total 22 nucleotides, A:4, T:7, G:5, C:6 (GC% 50)
	Wild M. Tb DNA (MPT64) or W-plasmid	TqMn-W-VIC: TaqMan probe-wild-VIC	5′-VIC-TATCGAT AGCGCCGAATGCCGG-TAMRA-3′ Total 22 nucleotides, A:5, T:4, G:7, C:6 (GC% 59)
	New internal Control (NM-plasmid)	TqMn-M-FAM: TaqMan probe- mutation-FAM	5′-FAM-ATGGGACGGCTAGCAATCCGTC-TAMRA-3′ Total 22 nucleotides, A:5, T:4, G:7, C:6 (GC% 59)

Underline: artificial sequence.