TABLE 2.
Conditions for multiplex PCRs of 12 MIRU-VNTR loci
| Multiplex | Locusa | VNTR length (bp) | MgCl2 concn (mM) | PCR primer pairs (5′ to 3′, with labeling indicated)b | Reference |
|---|---|---|---|---|---|
| Mix A | VNTR 0580 | 77 | 3.0 | GCGCGAGAGCCCGAACTGC (FAM) | 30 |
| GCGCAGCAGAAACGTCAGC | |||||
| VNTR 2996 | 51 | 3.0 | TAGGTCTACCGTCGAAATCTGTGAC | 30 | |
| CATAGGCGACCAGGCGAATAG (VIC) | |||||
| VNTR 0802 | 54 | 3.0 | GGGTTGCTGGATGACAACGTGT (NED) | 30 | |
| GGGTGATCTCGGCGAAATCAGATA | |||||
| Mix B | VNTR 0960 | 53 | 2.0 | GTTCTTGACCAACTGCAGTCGTCC | 30 |
| GCCACCTTGGTGATCAGCTACCT (FAM) | |||||
| VNTR 1644 | 53 | 2.0 | TCGGTGATCGGGTCCAGTCCAAGTA | 30 | |
| CCCGTCGTGCAGCCCTGGTAC (VIC) | |||||
| VNTR 3192 | 53 | 2.0 | ACTGATTGGCTTCATACGGCTTTA | 30 | |
| GTGCCGACGTGGTCTTGAT (NED) | |||||
| Mix F | VNTR 0424 | 51 | 1.5 | CTTGGCCGGCATCAAGCGCATTATT | 38 |
| GGCAGCAGAGCCCGGGATTCTTC (FAM) | |||||
| VNTR 0577 | 58 | 1.5 | CGAGAGTGGCAGTGGCGGTTATCT (VIC) | 38 | |
| AATGACTTGAACGCGCAAATTGTGA | |||||
| Mix G | VNTR 2401 | 58 | 3.0 | CTTGAAGCCCCGGTCTCATCTGT (FAM) | 38 |
| ACTTGAACCCCCACGCCCATTAGTA | |||||
| VNTR 3690 | 58 | 3.0 | CGGTGGAGGCGATGAACGTCTTC (VIC) | 38 | |
| TAGAGCGGCACGGGGGAAAGCTTAG | |||||
| VNTR 4156 | 59 | 3.0 | TGACCACGGATTGCTCTAGT | 38 | |
| GCCGGCGTCCATGTT (NED) | |||||
| Individual | VNTR 1982 | 78 | 1.5 | CCGGAATCTGCAATGGCGGCAAATTAAAAG | 26 |
| TGATCTGACTCTGCCGCCGCTGCAAATA (FAM) |
a
Locus designation according to the position (in kilobase pairs) on the M. tuberculosis H37Rv chromosome. VNTR 00580, 2996, 0802, 0960, 1644, and 3192 correspond to MIRU locus 4 (alias ETRD), 26, 40, 10, 16, and 31 (alias ETRE), respectively (12, 29). VNTR 577, 1982, and 4156 correspond to ETRC, QUB-18, and QUB 4156, respectively (12, 20, 24).
b
FAM, 6-carboxyfluorescein.