Journal of Isfahan Medical School

Journal of Isfahan Medical School

The Study of PncA Gene Using PCR-RFLP and Allele-Specific PCR Methods in Distinguishing Mycobacterium Bovis from Mycobacterium Tuberculosis

Document Type : Original Article (s)

Authors
Mohammad Reza Allahyar Torkaman 1
Fatemeh Maryam Sheikholslami 2
Parisa Farnia 3
Mohammad Hassan Shahhosseiny 4
Mohadeseh Mozafari 2
Mehdi Shamsi 2
Mohammad Reza Masjedi 5
Ali Akbar Velayati 5
1 Department of Biology, Sciences and Research Branch, Islamic Azad University and Mycobacteriology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
2 Mycobacteriology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3 Associate Professor, Mycobacteriology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Associate Professor, Department of Microbiology; Shahryar Branch; Islamic Azad University, Tehran, Iran
5 Professor, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran
Abstract
Background: Bovine tuberculosis is a chronic bacterial disease of cattle that occasionally affects human. A unique mutation at position 169 (C→G) in Mycobacterium bovis differentiates it from Mycobacterium tuberculosis. For the first time, PCR-RFLP method was used in this study to separate the two organisms.Methods: In this study, 98 tuberculosis patients were assessed using microscopic evaluations, culture and antibiogram. Then, nucleotide changes in position 169 were investigated using Allele-specific PCR and PCR-RFLP methods. Finally, the results were compared with spoligotyping results   .Findings: Antibiogram revealed 28 subjects (28.57%) to be resistant and 54 (55.1%) to be sensitive to pyrazinamide while and 16 cases (16.23%) showed no growth. Of 28 resistant patients, 3 (3.1%) were diagnosed with M. Bovis using the classical methods. Using molecular methods, 95 (96.93%) out of 98 subjects were diagnosed with M. tuberculosis and 3 (3.06%) with M. bovis. Allele-specific PCR and PCR-RFLP matched with spoligotyping results. Conclusion: Our result showed the incidence of M. bovis infection to be higher in Iranian population (3.1%) than other studied cases (0.5-1%). In addition, we showed that Allele-specific PCR is the method of choice because it is faster and cheaper than PCR-RFLP. However, PCR-RFLP could be a proper alternative to prevent probable mismatches in Allele-specific PCR.
Keywords
Mycobacterium tuberculosis
Mycobacterium bovis
Allele-specific PCR
PCR-RFLP
pncA
  1. Somoskovi A, Dormandy J, Parsons LM, Kaswa M, Goh KS, Rastogi N, et al. Sequencing of the pncA gene in members of the Mycobacterium tuberculosis complex has important diagnostic applications: Identification of a species-specific pncA mutation in "Mycobacterium canettii" and the reliable and rapid predictor of pyrazinamide resistance. J Clin Microbiol 2007; 45(2): 595-9.
  2. Mostowy S, Cousins D, Behr MA. Genomic interrogation of the dassie bacillus reveals it as a unique RD1 mutant within the Mycobacterium tuberculosis complex. J Bacteriol 2004; 186(1): 104-9.
  3. Barouni AS, Augusto CJ, Lopes MT, Zanini MS, Salas CE. A pncA polymorphism to differentiate between Mycobacterium bovis and Mycobacterium tuberculosis. Mol Cell Probes 2004; 18(3): 167-70.
  4. Majoor CJ, Magis-Escurra C, van IJ, Boeree MJ, van SD. Epidemiology of Mycobacterium bovis disease in humans, The Netherlands, 1993-2007. Emerg Infect Dis 2011; 17(3): 457-63.
  5. Scorpio A, Lindholm-Levy P, Heifets L, Gilman R, Siddiqi S, Cynamon M, et al. Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 1997; 41(3): 540-3.
  6. Jureen P, Werngren J, Toro JC, Hoffner S. Pyrazinamide resistance and pncA gene mutations in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2008; 52(5): 1852-4.
  7. Sun Z, Scorpio A, Zhang Y. The pncA gene from naturally pyrazinamide-resistant Mycobacterium avium encodes pyrazinamidase and confers pyrazinamide susceptibility to resistant M. tuberculosis complex organisms. Microbiology 1997; 143(Pt 10): 3367-73.
  8. Lee KW, Lee JM, Jung KS. Characterization of pncA mutations of pyrazinamide-resistant Mycobacterium tuberculosis in Korea. J Korean Med Sci 2001; 16(5): 537-43.
  9. Sekiguchi J, Nakamura T, Miyoshi-Akiyama T, Kirikae F, Kobayashi I, Augustynowicz-Kopec E, et al. Development and evaluation of a line probe assay for rapid identification of pncA mutations in pyrazinamide-resistant mycobacterium tuberculosis strains. J Clin Microbiol 2007; 45(9): 2802-7.
  10. Shenai S, Rodrigues C, Sadani M, Sukhadia N, Mehta A. Comparison of phenotypic and genotypic methods for pyrazinamide susceptibility testing. Indian J Tuberc 2009; 56(2): 82-90.
  11. Barco P, Cardoso RF, Hirata RD, Leite CQ, Pandolfi JR, Sato DN, et al. pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates from the southeast region of Brazil. J Antimicrob Chemother 2006; 58(5): 930-5.
  12. Espinosa de los Monteros LE, Galan JC, Gutierrez M, Samper S, Garcia Marin JF, Martin C, et al. Allele-specific PCR method based on pncA and oxyR sequences for distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: intraspecific M. bovis pncA sequence polymorphism. J Clin Microbiol 1998; 36(1): 239-42.
  13. Taylor GM, Goyal M, Legge AJ, Shaw RJ, Young D. Genotypic analysis of Mycobacterium tuberculosis from medieval human remains. Microbiology 1999; 145(Pt 4): 899-904.
  14. Herrera EA, Segovia M. Evaluation of mtp40 genomic fragment amplification for specific detection of Mycobacterium tuberculosis in clinical specimens. J Clin Microbiol 1996; 34(5): 1108-13.
  15. Weil A, Plikaytis BB, Butler WR, Woodley CL, Shinnick TM. The mtp40 gene is not present in all strains of Mycobacterium tuberculosis. J Clin Microbiol 1996; 34(9): 2309-11.
  16. Yuksel P, Tansel O. Characterization of pncA mutations of pyrazinamide-resistant Mycobac-terium tuberculosis in Turkey. New Microbiol 2009; 32(2): 153-8.
  17. Scorpio A, Collins D, Whipple D, Cave D, Bates J, Zhang Y. Rapid differentiation of bovine and human tubercle bacilli based on a characteristic mutation in the bovine pyrazinamidase gene. J Clin Microbiol 1997; 35(1): 106-10.
  18. Amirmozafari N, Ramezanzadeh R, Farnia P, Ghazi F. The Frequency of Beijing Genotype of Mycobacterium Tuberculosis Isolated from Tuberculosis Patients. rjms 2006; 13(52): 7-17.
  19. Farnia P, Varahram M, Doraghi M, et al. Druge Susceptibility Test Againt Mycobacterium Tuberculosis. Tehran: Mycobacteriology Research Center (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD); 2007.
  20. Miyata M, Barreto Santos AC, Mendes NH, Cunha EA, Melo FA, Leite CQ. Assessment of the quality of dna extracted by two techniques from Mycobacterium tuberculosis for fast molecular identification and genotyping. Braz J Microbiol 2011; 42(2): 774-7.
  21. Kamerbeek J, Schouls L, Kolk A, van AM, van SD, Kuijper S, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 1997; 35(4): 907-14.
  22. Kazempour M, Masjedi MR, Velayati AA, Tajeddin E, Farnia P, Kargar M, et al. Identification of mycobacterium tuberculosis beijing genotype using three different molecular methods. Koomesh 2009; 11(1): 7-14.
  23. Velayati AA, Farnia P, Mirsaeidi M, Reza MM. The most prevalent Mycobacterium tuberculosis superfamilies among Iranian and Afghan TB cases. Scand J Infect Dis 2006; 38(6-7): 463-8.
  24. Rohani M, Farnia P, Nasab MN, Moniri R, Torfeh M, Amiri MM. Beijing genotype and other predominant Mycobacterium tuberculosis spoligotypes observed in Mashhad city, Iran. Indian J Med Microbiol 2009; 27(4): 306-10.
  25. de Kantor IN, Ambroggi M, Poggi S, Morcillo N, Da Silva Telles MA, Osorio RM, et al. Human Mycobacterium bovis infection in ten Latin American countries. Tuberculosis (Edinb) 2008; 88(4): 358-65.
  26. Pavlik I, Yayo Ayele W, Havelkova M, Svejnochova M, Katalinic-Jankovi V. Mycobacterium bovis in human population in four. Central European countries during 1990–1999. Vet Med - Czech 2003; 48(4): 90-8.
  27. Velayati AA, Farnia P, Boloorsaze MR, Sheikholslami MF, Khalilzadeh S, Hakeeme SS, et al. Mycobacterium bovis infection in children in the same family: transmission through inhalation. Monaldi Arch Chest Dis 2007; 67(3): 169-72.
Journal of Isfahan Medical School
Volume 29, Issue 157 - Serial Number 157
September and October 2011
Pages 1532-1541

  • Receive Date 12 July 2011
  • Accept Date 06 April 2022