PCR-SSCCP analysis in detecting point mutations targeting rpoB, katG and inhA genes for determining multi-drug resistance in Mycobacterium bovis and Mycobacterium tuberculosis strains
284 / 123
Authors
-
ALEX KIDANGAN
Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh 243 122 India
-
RISHENDRA VERMA
Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh 243 122 India
Keywords:
Mycobacterium bovis, M. tuberculosis, PCR-SSCPAbstract
The usefulness of polymerase chain reaction-single stranded confirmation polymorphism (PCR-SSCP) for determination of rifampicin and isoniazid resistance in Mycobacterium tuberculosis and M. bovis cultures from human and animal origin was investigated. Mycobacteria (81) in the study included, viz. 12 MDR-TB samples, 35 sputum samples, 3 lung and lymphnode tissues from bovines and 11 M. tuberculosis and 18 M. bovis culture, M. tuberculosis H37Rv and M. bovis BCG strain). All the mycobacterial cultures were characterized on growth characteristics, biochemical test pattern, MTB complex specific IS6110 PCR and species specific 12.7 kb multiplex PCR. PCR-SSCP was used to determine resistance against rifiampin by targeting rpoB gene (305 bp) and isoniazid by targeting katG (237 bp) and inhA (261 bp). Rifampicin resistance was detected by PCR-SSCP in 1 out of 12 MDR-TB samples (8.3%), while isoniazid resistance was detected in 66.7% of MDR-TB samples using PCRSSCP of katG and 75% of MDR-TB samples using inhA SSCP analysis.Downloads
References
Banerjee A, Dubnau E, Quemard A, Balasubramanian V, Um K S, Wilson T, Collins D, de Lisle G and Jacobs Jr W R. 1994. inhA, a gene encoding a target for INH and ethionamide in Mycobacterium tuberculosis. Science 263: 227–30. DOI: https://doi.org/10.1126/science.8284673
Bassam B J, Caetano-Anolles G and Gresshoff P M. 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Analytical Biochemistry 196 (1): 80–83. DOI: https://doi.org/10.1016/0003-2697(91)90120-I
Chakravorty S and Tyagi J S. 2005. Novel multipurpose methodology for detection of mycobacteria in pulmonary and extrapulmonary specimens by smear microscopy, culture, and PCR. Journal of Clinical Microbiology 43: 2697– 702. DOI: https://doi.org/10.1128/JCM.43.6.2697-2702.2005
Chakravorty S, Dudeja M, Hanif M and Tyagi J S. 2005. Utility of USP smear microscopy, culture and PCR in the diagnosis of pulmonary tuberculosis. Journal of Clinical Microbiology 43: 2703–08. DOI: https://doi.org/10.1128/JCM.43.6.2703-2708.2005
Coleman JD and Cooke MM. 2001. Mycobacterium bovis infection in wildlife in New Zealand. Tuberculosis (Edinburgh) 81:191–202. DOI: https://doi.org/10.1054/tube.2001.0291
Honore N and Cole ST. 1993. Molecular basis of Rifampin resistance in Mycobacterium leprae. Antimicrobial Agents Chemotherapy 37:414–18. DOI: https://doi.org/10.1128/AAC.37.3.414
Heym B, Honore N, Schurra C, Cole ST, Heym B, Truffot-Pernot C, Grosset JH, Banerjee A, Jacobs Jr WR and van Embden JDA. 1994. Implications of multidrug resistance for the future of short course chemotherapy of tuberculosis: a molecular study. Lancet 344: 293–98. DOI: https://doi.org/10.1016/S0140-6736(94)91338-2
Miller LP, Crawford JT and Shinnick TM. 1994 The rpoB gene of Mycobacterium tuberculosis. Antimicrobial Agents Chemotherapy 38: 805–11. DOI: https://doi.org/10.1128/AAC.38.4.805
Jin DJ and Gross CA. 1988 Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance. Journal of Molecular Biology 202: 45–48. DOI: https://doi.org/10.1016/0022-2836(88)90517-7
Kapur V, Li LL, Iordanescu S, Hamrick MR, Wanger A, Kreiswirth BN and Musser MJ. 1994. Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNApolymerase sub-unit in rifampin resistant Mycobacterium tuberculosis strains from New York city and Texas. Journal of Clinical Microbiology 32: 1095–98. DOI: https://doi.org/10.1128/jcm.32.4.1095-1098.1994
Lisitsyn NA, Sverdlov EV, Moiseyeva EP, Danilevskaya ON and Nikiforov VG. 1984. Mutation to rifampin resistance at the beginning of the RNA polymerase beta subunit gene in Escherichia coli. Molecular and General Genetics 196:173– 174. DOI: https://doi.org/10.1007/BF00334112
Ovchinnikov YA, Monastyrskaya GS, Guriev SO, Kalinina NF, Sverdlov ED, Gragerov AI, Bass IA, Kiver IF, Moiseyeva EP, Igumnov VN, Mindlin SZ, Nikiforov VG and Khesin RB. 1983. RNA polymerase rifampicin resistance mutations in Escherichia coli: sequence changes and dominance. Molecular and General Genetics 190: 344–48. DOI: https://doi.org/10.1007/BF00330662
Marks J. 1972. Classification of the mycobacteria in relation to clinical significance. Tubercle, 53: 259. DOI: https://doi.org/10.1016/0041-3879(72)90004-9
Petroff SA. 1915. A new and rapid method for the isolation and cultivation of tubercle bacilli directly from the sputum and feces. The Journal of Experimental Medicine 21: 38–42. DOI: https://doi.org/10.1084/jem.21.1.38
Sechi LA, Sanguinetti M, Molicotti PL Romano, Leori G, Delogu G, Boccia S, M La Sorda and Fadda G. 2001. Molecular basis of rifampin and isoniazid resistance in Mycobacterium bovis strains isolated in Sardinia Itlay. Antimicrob Agents Chemother. 45(6): 1645–48. DOI: https://doi.org/10.1128/AAC.45.6.1645-1648.2001
Silva MSN, Senna SG, Ribeiro MO, Valim ARM, Telles MA, Kritski A, Morlock GP, Cooksey RC, Zaha A and Rossetti MLR. 2003. Mutations in katG, inhA and ahpC genes of Brazilian Isoniazid resistant isolates of Mycobacterium tuberculosis. Journal of Clinical Microbiology 41(9): 4471– 74. DOI: https://doi.org/10.1128/JCM.41.9.4471-4474.2003
Telenti A, Imboden P, Marchesi F, Schmedheim T and Bodmer T. 1993. Direct automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single stranded conformation polymorphism analysis. Antimicrobial Agents Chemotherapy 37: 2054–58. DOI: https://doi.org/10.1128/AAC.37.10.2054
Thangaselvam M, Kidangan A, Verma Rishendra and Ramane SP. 2009. Molecular Detection and Differentiation of Mycobacterium tuberculosis Complex in Human Sputum Samples Using PCR Assays: A Preliminary Report. Indian Journal of Veterinary Research 18(2):50–54.
Victor T, Du Toit R and van Helden PD. 1992. Purification of sputum samples through Sucrose improves detection of Mycobacterium tuberculosis by Polymerase Chain Reaction. Journal of Clinical Microbiology 30(6): 1514–17. DOI: https://doi.org/10.1128/jcm.30.6.1514-1517.1992
Williams DL, Waguespack C, Eisenach K, Crawford JT, Portaels F, Salfinger M, Nolan CM, Abe C, Sticht-Groh V and Gillis TP. 1994. Characterization of Rifampin resistance in pathogenic Mycobacteria. Antimicrobial agents and Chemotherapy 38(10): 2380–86. DOI: https://doi.org/10.1128/AAC.38.10.2380
Zhang Y, Heym B, Young D and Cole S. 1992. The catalase- peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature 358: 591–93. DOI: https://doi.org/10.1038/358591a0
Downloads
Submitted
Published
Issue
Section
License
Copyright (c) 2014 The Indian Journal of Animal Sciences
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
The copyright of the articles published in The Indian Journal of Animal Sciences is vested with the Indian Council of Agricultural Research, which reserves the right to enter into any agreement with any organization in India or abroad, for reprography, photocopying, storage and dissemination of information. The Council has no objection to using the material, provided the information is not being utilized for commercial purposes and wherever the information is being used, proper credit is given to ICAR.
How to Cite
