Article Figures & Data
Figures
- FIG. 1.
SSCP analysis of katG315, inhA (promoter and structural ORFs), oxyR-ahpC intergenic region, and three regions of kasA in INH-resistant M. tuberculosis. All PCR products were denatured in the presence of 95% deionized formamide and electrophoresed by using specific conditions shown in Table 2 and by using a GenePhor electrophoresis system (Amersham Biosciences). (A) katG315 (145-bp PCR products). Lanes: 3 and 10, wild-type control (H37Rv); 1, W341S; 2, S315R; 4 and 6, S315N; 7, S315G; 5 and 8, wild type (susceptible isolates); and 9, S315T. (B) inhA promoter (187-bp PCR products). Lanes: 1, wild-type control (H37Rv); 2 and 4, C-17T; 3, C-15T. (C) inhA structural gene (codon 16) (222-bp PCR products). Lanes: 1 and 6, wild-type control (H37Rv); 2, I21V; 3, wild type (susceptible isolate); 4, L44L; 5, I21T. (D) oxyR-ahpC intergenic region (264-bp PCR products). Lanes: 1, 7, and 13, wild-type control (H37Rv); 2, oxyR; 5, C−15T; 6, C−39T; 8, C−10T; 9, G−48A; 10, C−12T; 11, G-9A; 12, C-39T; 3, and 4, wild-type (susceptible isolates). (E) kasA region 3 (269-bp PCR products). Lanes: 1, wild-type control (H37Rv); 2, 4, and 5, H180H; 3, G149G; and 6, wild type (susceptible isolates). (F) kasA region 4 (263-bp PCR products). Lanes: 1, wild-type control (H37Rv); 2 and 3, G269S. (G) kasA region 5 (223-bp PCR products). Lanes: 1, wild-type control (H37Rv); 2, G318G; and 3, wild type (susceptible isolates).
Tables
- TABLE 1.
Oligonucleotide primers used for PCR-SSCP analyses of katG, inhA (regulatory and structural regions), kasA, and the oxyR-ahpC intergenic region
Accession no. Primer Primer sequence (5′ to 3′) Nucleotide locationa PCR annealing temp (°C) PCR product size (bp) Z97193 katG-315F AGA GCT CGT ATG GCA CCG GA 32059 58 145 katG-315R CCA GCA GGG CTC TTC GTC AG 31915 katG-463F CTG CTG TGG CAG GAT CCG GT 31636 60 162 katG-463R GCG CTT GTC GCT ACC ACG GA 31494 V66801 inhA-1F GCT GAG TCA CAC CGA CAA ACG 909 60 187 inhA-1R CCA GGA CTG AAC GGG ATA CGA 1075 V02492 inhA-16F TGA CAC AAC ACA AGG ACG CA 1779 52 222 inhA-16R GTT TTG CAC GTC GAG TTC GA 1981 AF106077 inhA-94F GCA AAA CGA GGA GCA CCT GGC 1994 63 182 inhA-94R AAT ACG CCG AGA TGT GGA TGC 2156 V16243 ahpC-F CTT GCG GCA CTG CTG AAC CAC 7147 60 264 ahpC-R ACA GGT CAC CGC CGA TGA GAG 7389 Z70692 kasA-1F AAC GTT CAG GCG AGG CTT GA 30631 68 262 kasA-1R ATG TCG AGT CGG CCC ATG TG 30873 kasA-2F CGG TCA CCT CAA GGA TCC GG 30844 58 267 kasA-2R GCA CCG TTG GGC ATG ATC AT 31092 kasA-3F GGA AGG TGT CCC CGC TGG CC 31066 63 269 kasA-3R TCA GGC TCG TCG TTG CGG GT 31314 kasA-4F TTC TCC ATG ATG CGG GCC AT 31290 68 263 kasA-4R AGC TCC AGC GAG CGA GTC AT 31533 kasA-5F CCG ATG GTG TTC GTG CCG GT 31507 63 223 kasA-5R ACC GAC TCG AGC GCA CCG AC 31710 kasA-6F GTC TGC GTC GGG CCA CTC GA 31682 63 255 kasA-6R CTC GCG GCG ACC CGC GAT GTC 31917 ↵ a Numbers refer to the nucleotide position within the referenced GenBank sequence of the 5′ primer terminus.
- TABLE 2.
Conditions used for SSCP electrophoresisa
Primer pair(s) (forward/reverse) Volts Temp (°C) katG-315F/315R 350 12 katG-463F/463R 400 8 inhA-F/1R 350 12 inhA-16/17R 350 12 inhA-94/94R 400 12 ahpC-F/ahpC R 400 12 kasA-F/3R and -5F/5R 600 13 kasA-2F/2R and -6F/6R 450 8 kasA-1F/1R and -4F/4R 500 13 ↵ a GenePhor electrophoresis system using GeneGel Excel 12.5/24 gel (Amersham Biosciences). All PCR amplicons were run for 2 h under the indicated conditions.
- TABLE 3.
Oligonucleotide primers used for PCR and the sequencing reaction of katG, inhA (regulatory and structural), the oxyR-ahpC intergenic region, and kasA
Accession no. Primer Primer sequence (5′ to 3′) Nucleotide locationa PCR annealing temp (°C) PCR product size (bp) U41314 BC 48 TGG CCG CGG CGG TCG ACA TT 725 58 322 BC 51R CCA GCA GGG CTC TTC GTC AG 1027 Z79701 InhA-1 CCT CGC TGC CCA GAA AGG GA 870 60 248 InhA-2 ATC CCC CGG TTT CCT CCG GT 1098 InhA-5 TGG ACG GCA AAC GGA TTC TGG 1813 60 366 InhA-6 ACG AAT ACG CCG AGA TGT GGA 2158 Z81451 AhpC-1 GCC TGG GTG TTC GTC ACT GGT 7455 60 359 AhpC-2 CGC AAC GTC GAC TGG CTC ATA 7117 Z70692 KasA-1 CGT TCA GGC GAG GCT TGA GGC 93 68 700 KasA-5 CAG GCT CGT CGT TGC GGG TC 773 KasA-4 TGC CCA TCG CGG CGT TCT CCA 736 68 703 KasA-8 GTC CGA CTC GCC CCC GCA AGC 1418 KasA-2 CCA GAC CGG TTC GCC GTT GTT 435 KasA-6 CGC CGG CGG ACA TCG ACC AC 1024 KasA-7 GCC GTG CCG TGC GCG TTG A 1045 X68081 KatG-1 GCC CGA TAA CAC CAA CTC CTG 1947 68 680 KatG-5 CAG ATC CCG CTA CCG CTG TA 2606 KatG-2 CGC CGA CTA CGG CCA CTA 2254 KatG-3 GCC ACG CCA TCC GGA TAA 2238 KatG-4 CCT GGC TCG GCG ATG A 2586 68 648 KatG-9 CTC GGT GGA TCA GCT TGT ACC 3213 KatG-6 CTC GAT GGC ACC GGA ACC 2884 KatG-7 CGT CGG GGT GTT CGT CCA TAC 2936 KatG-8 GAG GAA TTG GCC GAC GAG TT 3182 68 667 KatG-13 TCT CAG GGG CAC TGA GCG TAA 3828 KatG-10 CAA GTC GGG TGG GAG GTC AA 3482 KatG-11 CTC TTC CAG GGT GCG AAT GAC 3527 KatG-12 GCC GAG TAC ATG CTG CTC GAC 3794 60 452 KatG-14 CGG CGG GTT GTG GTT GA 4229 ↵ a Numbers refer to the nucleotide position within the referenced GenBank sequence of the 5′ primer terminus. Oligonucleotides KasA-2, KasA-6, KasA-7, KatG-2, KatG-3, KatG-6, KatG-7, KatG-10, and KatG-11 were used only for sequencing reactions.
- TABLE 4.
Mutations found in katG, kasA, inhA, and the ahpC regulatory region in 97 INH-resistant M. tuberculosis isolates
Gene No. of isolates Mutationa INH MICs in μg/ml (no. of isolates if >1) Additional mutation(s) Nucleotide no. Amino acid katG 1 A inserted at position 17 Frameshift* 32 1 T→C at position 271 W91R* >32 1 A→G at position 290 H97R* 8 katG 1 C→T at position 326 A109V* 16 oxyR-ahpC 1 C→T at position 329 A110A* 16 katG, inhA P 1 C→T at position 431 A144V* 1 2 A→G at position 598 K200E* 8, >32 katG, inhA P, oxyR-ahpC 1 C→G at position 695 P232R* 32 inhA P 1 G→T at position 761 R254L* NG kasA 1 G→T at position 817 G273C* >32 oxyR-ahpC 1 G→A at position 836 G279D* >32 inhA P, oxyR-ahpC 1 C→G at position 877 L293V* 16 inhA P 1 G→A at position 895 G299S* >32 oxyR-ahpC, kasA 1 A→C at position 904 S302R 8 oxyR-ahpC 1 A→G at position 943 S315G 8 inhA P 1 A→C at position 943 S315R >32 49 G→C at position 944 S315T 4 (23), 8 (15), 16 (5), 32 (3), >32 (3) inhA P, kasA, katG 7 G→A at position 944 S315N 4 (3), 16, >32 (3) inhA P, katG 1 G→T at position 944 S315I 16 1 C→G at position 945 S315R 32 1 G→C at position 1022 W341S* >32 inhA P 1 G→T at position 1255 D419Y* 16 inhA P 2 A→C at position 1256 D419A* 2, 8 InhA P 1 C→A at position 1257 D419E* >32 katG, inhA, oxyR-ahpC 1 T insertion at position 1311 Frameshift* >32 oxyR-ahpC 1 A insertion at position 1329 Frameshift* 16 katG 1 C del at position 1339 Frameshift* 16 katG 2 G→T at position 1388 R463L >32 (2) katG 1 G→A at position 1468 G490S* 16 1 C→T at position 1833 L611L 4 inhA P 1 Deletion after position 1845 NA* >32 oxyR-ahpC, kasA 1 T→G at position 1985 L662R* 32 1 C deletion at position 2139 Frameshift* >32 oxyR-ahpC 1 G→A at position 2176 A726T* 16 inhA P inhA promoter (P) 23 C→T at position −15 NA 1, 2 (3), 4, 8 (6), 16 (5), 32, >32 (6) katG, kasA, oxyR-ahpC, inhA 2 G→T at position −17 NA 16, 32 katG inhA structural 1 A→G at position 61 I21V 16 katG, kasA 4 T→C at position 62 I21T 8, 16 (2), >32 InhA P 1 C→T at position 130 L44L >32 katG, inhA P, oxyR-ahpC oxyR-ahpC 2 G→A at position −9 NA 16, >32 katG 1 C→T at position −10 NA >32 katG, kasA 1 C→T at position −12 NA >32 katG 1 C→T at position −15 NA >32 katG 4 C→T at position −39** NA 8, >32 (3) katG, kasA 1 G→A at position −48* NA >32 katG ahpC 1 T→C at position 30 I10I* 4 kasA 13 G→A at position 805 G269S 1, 4 (3), 8 (3), 16 (2), >32 (3), NGb katG, inhA P, oxyR-ahpC, inhA, kasA
