Optimal DNA Isolation Method for Detection of Nontuberculous Mycobacteria by Polymerase Chain Reaction

Authors

1 Department of Microbiology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

2 Department of Medical Parasitology and Mycology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

3 Regional Tuberculosis Reference Laboratories in Isfahan, Isfahan, Iran

Abstract

Background: Nontuberculous mycobacteria (NTM) are a group of opportunistic pathogens and these are widely dispersed in water and soil resources. Identification of mycobacteria isolates by conventional methods including biochemical tests, growth rates, colony pigmentation, and presence of acid-fast bacilli is widely used, but these methods are time-consuming, labor-intensive, and may sometimes remain inconclusive. Materials and Methods: The DNA was extracted from NTM cultures using CTAB, Chelex, Chelex + Nonidet P-40, FTA® Elute card, and boiling The quantity and quality of the DNA extracted via these methods were determined using UV-photometer at 260 and 280 nm, and polymerase chain reaction (PCR) amplification of the heat-shock protein 65 gene with serially diluted DNA samples. Results: The CTAB method showed more positive results at 1:10–1:100,000 at which the DNA amount was substantial. With the Chelex method of DNA extraction, PCR amplification was detected at 1:10 and 1:1000 dilutions. Conclusions: According to the electrophoresis results, the CTAB and Chelex DNA extraction methods were more successful in comparison with the others as regard producing suitable concentrations of DNA with the minimum use of PCR inhibitor.

Keywords

1.
Crago B, Ferrato C, Drews SJ, Louie T, Ceri H, Turner RJ, et al. Surveillance and molecular characterization of non-tuberculous mycobacteria in a hospital water distribution system over a three-year period. J Hosp Infect 2014;87:59-62.  Back to cited text no. 1
[PUBMED]    
2.
Tortoli E. Microbiological features and clinical relevance of new species of the genus Mycobacterium. Clin Microbiol Rev 2014;27:727-52.  Back to cited text no. 2
[PUBMED]    
3.
van der Werf MJ, Ködmön C, Katalinic-Jankovic V, Kummik T, Soini H, Richter E, et al. Inventory study of non-tuberculous mycobacteria in the European Union. BMC Infect Dis 2014;14:62.  Back to cited text no. 3
    
4.
Katoch VM. Infections due to non-tuberculous mycobacteria (NTM). Indian J Med Res 2004;120:290-304.  Back to cited text no. 4
[PUBMED]    
5.
Mokaddas E, Ahmad S. Development and evaluation of a multiplex PCR for rapid detection and differentiation of Mycobacterium tuberculosis complex members from non-tuberculous mycobacteria. Jpn J Infect Dis 2007;60:140-4.  Back to cited text no. 5
[PUBMED]    
6.
Nasr-Esfahani B, Sarikhani E, Moghim S, Faghri J, Fazeli H, Hoseini N, et al. Molecular characterization of environmental non-tuberculous mycobacteria using PCR – RFLP analysis of 441 Bp heat shock protein 65 fragments. Iran J Public Health 2012;41:108-14.  Back to cited text no. 6
[PUBMED]    
7.
Serkani JE, Isfahani BN, Safaei HG, Kermanshahi RK, Asghari G. Evaluation of the effect of Humulus lupulus alcoholic extract on rifampin-sensitive and resistant isolates of Mycobacterium tuberculosis. Res Pharm Sci 2012;7:235-42.  Back to cited text no. 7
[PUBMED]    
8.
Covert TC, Rodgers MR, Reyes AL, Stelma GN Jr. Occurrence of nontuberculous mycobacteria in environmental samples. Appl Environ Microbiol 1999;65:2492-6.  Back to cited text no. 8
[PUBMED]    
9.
Soini H, Musser JM. Molecular diagnosis of mycobacteria. Clin Chem 2001;47:809-14.  Back to cited text no. 9
[PUBMED]    
10.
Timms VJ, Mitchell HM, Neilan BA. Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis. J Microbiol Methods 2015;112:99-103.  Back to cited text no. 10
[PUBMED]    
11.
Hadifar S, Moghim S, Fazeli H, GhasemianSafaei H, Havaei SA, Farid F, et al. Molecular typing of Iranian mycobacteria isolates by polymerase chain reaction-restriction fragment length polymorphism analysis of 360-bp rpoB gene. Adv Biomed Res 2015;4:152.  Back to cited text no. 11
[PUBMED]    
12.
Nasr Esfahani B, Rezaei Yazdi H, Moghim S, Ghasemian Safaei H, Zarkesh Esfahani H. Rapid and accurate identification of Mycobacterium tuberculosis complex and common non-tuberculous mycobacteria by multiplex real-time PCR targeting different housekeeping genes. Curr Microbiol 2012;65:493-9.  Back to cited text no. 12
[PUBMED]    
13.
Hosek J, Svastova P, Moravkova M, Pavlik I, Bartos M. Methods of mycobacterial DNA isolation from different biological material: A review. Vet Med 2006;51:180-92.  Back to cited text no. 13
    
14.
De Almeida IN, Da Silva Carvalho W, Rossetti ML, Costa ER, De Miranda SS. Evaluation of six different DNA extraction methods for detection of Mycobacterium tuberculosis by means of PCR-IS6110: Preliminary study. BMC Res Notes 2013;6:561.  Back to cited text no. 14
[PUBMED]    
15.
Amaro A, Duarte E, Amado A, Ferronha H, Botelho A. Comparison of three DNA extraction methods for Mycobacterium bovisMycobacterium tuberculosis and Mycobacterium avium subsp. avium. Lett Appl Microbiol 2008;47:8-11.  Back to cited text no. 15
[PUBMED]    
16.
Aldous WK, Pounder JI, Cloud JL, Woods GL. Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR. J Clin Microbiol 2005;43:2471-3.  Back to cited text no. 16
[PUBMED]    
17.
Amita J, Vandana T, Guleria R, Verma R. Qualitative evaluation of mycobacterial DNA extraction protocols for polymerase chain reaction. Mol Biol Today 2002;3:43-9.  Back to cited text no. 17
    
18.
Rastogi N, Goh KS, Berchel M. Species-specific identification of Mycobacterium leprae by PCR-restriction fragment length polymorphism analysis of the hsp65 gene. J Clin Microbiol 1999;37:2016-9.  Back to cited text no. 18
[PUBMED]    
19.
Aye KS, Matsuoka M, Kai M, Kyaw K, Win AA, Shwe MM, et al. FTA card utility for PCR detection of Mycobacterium leprae. Jpn J Infect Dis 2011;64:246-8.  Back to cited text no. 19
[PUBMED]    
20.
Steingrube VA, Gibson JL, Brown BA, Zhang Y, Wilson RW, Rajagopalan M, et al. PCR amplification and restriction endonuclease analysis of a 65-kilodalton heat shock protein gene sequence for taxonomic separation of rapidly growing mycobacteria. J Clin Microbiol 1995;33:149-53.  Back to cited text no. 20
[PUBMED]    
21.
Mirsaeidi M, Farnia P, Sadikot R, Hsueh PR, Aliberti S. Nontuberculous mycobacteria: Epidemiologic, mycobacteriologic, and clinical aspects. Biomed Res Int 2015;2015:523697.  Back to cited text no. 21
[PUBMED]    
22.
Sinha P, Gupta A, Prakash P, Anupurba S, Tripathi R, Srivastava GN. Differentiation of Mycobacterium tuberculosis complex from non-tubercular mycobacteria by nested multiplex PCR targeting IS6110, MTP40 and 32kD alpha antigen encoding gene fragments. BMC Infect Dis 2016;16:123.  Back to cited text no. 22
[PUBMED]    
23.
Leite FL, Stokes KD, Robbe-Austerman S, Stabel JR. Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction. J Vet Diagn Invest 2013;25:27-34.  Back to cited text no. 23
[PUBMED]    
24.
Mita A, Mori Y, Nakagawa T, Tasaki T, Utiyama K, Mori H. Comparison of fecal pooling methods and DNA extraction kits for the detection of Mycobacterium avium subspecies paratuberculosis. Microbiologyopen 2016;5:134-42.  Back to cited text no. 24
[PUBMED]    
25.
Nagdev KJ, Kashyap RS, Deshpande PS, Purohit HJ, Taori GM, Daginawala HF. Determination of polymerase chain reaction efficiency for diagnosis of tuberculous meningitis in Chelex-100 extracted DNA samples. Int J Tuberc Lung Dis 2010;14:1032-8.  Back to cited text no. 25
[PUBMED]    
26.
Santos A, Cremades R, Rodríguez JC, García-Pachón E, Ruiz M, Royo G. Comparison of methods of DNA extraction for real-time PCR in a model of pleural tuberculosis. APMIS 2010;118:60-5.  Back to cited text no. 26
    
27.
Polski JM, Kimzey S, Percival RW, Grosso LE. Rapid and effective processing of blood specimens for diagnostic PCR using filter paper and Chelex-100. Mol Pathol 1998;51:215-7.  Back to cited text no. 27
[PUBMED]    
28.
de Almeida IN, Aleixo AV, Carvalho Wda S, de Miranda SS. In-house PCR with DNA extracted directly from positive slides to confirm or exclude the diagnosis of tuberculosis: Focus on biosafety. Rev Argent Microbiol 2015;47:47-9.  Back to cited text no. 28
[PUBMED]    
29.
Jaravata CV, Smith WL, Rensen GJ, Ruzante J, Cullor JS. Survey of ground beef for the detection of Mycobacterium avium paratuberculosis. Foodborne Pathog Dis 2007;4:103-6.  Back to cited text no. 29
[PUBMED]    
30.
Gustavsson I, Lindell M, Wilander E, Strand A, Gyllensten U. Use of FTA card for dry collection, transportation and storage of cervical cell specimen to detect high-risk HPV. J Clin Virol 2009;46:112-6.  Back to cited text no. 30
[PUBMED]    
31.
Milne E, van Bockxmeer FM, Robertson L, Brisbane JM, Ashton LJ, Scott RJ, et al. Buccal DNA collection: Comparison of buccal swabs with FTA cards. Cancer Epidemiol Biomarkers Prev 2006;15:816-9.  Back to cited text no. 31
[PUBMED]    
32.
Hill EB, Wayne LG, Gross WM. Purification of mycobacterial deoxyribonucleic acid. J Bacteriol 1972;112:1033-9.  Back to cited text no. 32
[PUBMED]    
33.
Salgado M, Verdugo C, Heuer C, Castillo P, Zamorano P. A novel low-cost method for Mycobacterium avium subsp. paratuberculosis DNA extraction from an automated broth culture system for real-time PCR analysis. J Vet Sci 2014;15:233-9.  Back to cited text no. 33
[PUBMED]