| | 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) |
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