https://imcjms.com Ibrahim Medical College Journal of Medical Science https://imcjms.com/registration/journal_full_text/49 Tue, 02 Aug 2016 10:51:32 +0000 Sensitivity of the molecular diagnostic tests of extrapulmonary tuberculosis largely depends upon the efficiency of DNA extraction methods. The objective of our study was to compare three methods of extracting DNA of Mycobacterium tuberculosis for testing by polymerase chain reaction. All three methods; heating, heating with sonication and addition of lysis buffer with heating and sonication were implicated on 20 extrapulmonary samples. PCR positivity was 2 (10%), 4 (20%) and 7 (35%) in the samples extracted by heating, heat+sonication and heat+sonication+lysis buffer method respectively. Of the extraction methods evaluated, maximum PCR positive results were achieved by combined heat, sonication and lysis buffer method which can be applied in routine clinical practice. Address for Correspondence:Dr. Khandaker Shadia, Assistant Professor, Department of Microbiology, Ibrahim Medical College, 122 Kazi Nazrul Islam Avenue, Dhaka 1000, Bangladesh, e-mail: drsadialima@ymail.com   Isolation of nucleic acid (DNA) from mycobacteria is difficult due to its complex cell wall structure.1 Therefore, most of the simple and commonly used DNA extraction methods result in poor quality and low yield of DNA, which is also affected by type of sample used.2 However, the sensitivity of molecular diagnosis is largely dependent on the efficiency of cell lysis and extraction of DNA.3 Several methods for mycobacterial cell wall lysis and DNA extraction have been used like simple boiling in distilled water, disruption by glass bead or sonication, enzymatic lysis, chemical lysis or combination of these techniques.4 The objective of this study was to compare three methods of extracting M. tuberculosis DNA from various types of extrapulmonary specimens. Materials and Methods During DNA extraction in heat method aliquots of the sediments were washed with distilled water and boiled at 95°C for 30 minutes in a water bath. After centrifugation at 10,000 rpm for 5 minutes supernatant was collected in a 1.5 ml tube.7,8 In combined heat and sonication method, after removal from water bath lysate was sonicated in an ultrasonication bath (Branson 1200 E4, Branson Co, Danbury, CT) for 15 min at    30 W.9,10 Then supernatant was recovered in the same way. In the third method, at first deposit was suspended in 135 ml of lysis buffer [Prepared by mixing 20 mM Tris/HCl (pH 8.3), 1mg proteinase K/ml, 0.5% Tween 20 and 10ml sterile distilled water] instead of distilled water and incubated at 56°C for 3 hours. Then the lysate cooled to room temperature and centrifuged at 12000 rpm for 15 min. Resultant pellet was resuspended with 100 ml distilled water and rest of the method repeated as method-2. Briefly, heating, sonication, centrifugation and finally collection of the supernatant in 1.5 ml tube.11,12 Amplification and detection procedures   Three DNA extraction methods were applied in 8 AFB smear and/or culture positive and 12 negative samples. Maximum positivity was seen in the method using heat, sonication and lysis buffer in combination. Among 8 AFB smear and/or culture positive cases 6 (75%) were positive by this method whereas 4 (50%) were positive by heat-sonication and 2 (25%) by only heating method. Among 12 AFB smear and/or culture negative samples one (8.3%) sample was PCR positive by heat+ sonication+lysis buffer method. None of the AFB negative sample was positive by only heating or heat + sonication method. Table 1: Comparison of PCR results with the DNA extracted by three different methods (n=20).   Several DNA extraction methods can be employed for the isolation of mycobacterial nucleic acid from clinical samples. But in case of samples of extrapulmonary TB more precise method is required due to the paucibacillary nature of these samples. Moreover in highly TB prevalent country like Bangladesh the method should be simpler and reasonably cost effective. In this context, kit based extraction offers quality-controlled reagents with optimized compositions for all steps, but they are relatively costly and have variable sensitivity.14 Heating is simplest and widely used method but its sensitivity in smear negative pulmonary as well as extrapulmonary samples is not satisfactory.7,8,15 In fact, association of physical disintegration by bead beating or sonication in a specific enzyme and detergent containing buffer is appropriate for mycobacterial cell wall lysis.14  Though the main disadvantage of the sonication method was the need of a sonicator for cell lysis and the third method needs extra 3 hours incubation but in consideration of proper diagnosis it can be acceptable. This incubation time can be lowered by further experiment as different authors mentioned different ranges of incubation times.8,11,12 Lastly, from our experiment it is apparent that combination of lysis buffer and sonication with heating is considerably bring better DNA yield in detecting M. tuberculosis by PCR, especially in samples with extrapulmonary samples that have low number of mycobacteria. Obviously the limitation of our study is small sample size and lack of observation of quality and quantity of recovered DNA. Further study with large number of pulmonary and extrapulmonary samples following necessary modification may strengthen our findings. 1.   Barry MC, Mdluli K. Drug sensitivity and environmental adaptation of Mycobacterial cell wall components. Trends Microbiol 1996; 4: 275-8. 3.   Honore’-Bouakline S, Vincensini JP, Giacuzzo V et al. Rapid Diagnosis of Extrapulmonary Tuberculosis by PCR: Impact of Sample Preparation and DNA Extraction. J Clin Microbiol 2003; 2323-2329. 5.   Khaled NA and Enarson DA. Tuberculosis. A Manual for medical Students. Geneva, World Health Organization. (WHO/CDS/ TB/99.272) 1999; 14-21. 7.   Aldous WK, Pounder JI, Cloud JL and Woods GL. Comparison of Six Methods of Extracting Mycobacterium tuberculosis DNA from Processed Sputum for Testing by Quantitative. J Clin Microbiol 2005; 43(5): 2471–73. 9.   Buck GE, O’Hara LC & Summersgill JT. Rapid simple method for treating clinical specimens containing Mycobacterium tuberculosis to remove DNA for polymerase chain reaction. J Clin Microbiol 1992; 30: 1331–1334. 11.Orallo RLC, Mendoza MT, Ann MD et al. Evaluation of the Usefulness of PCR in the diagnosis of Mycobacterium tuberculosis in tissues and body fluids in UP-Philippine General Hospital. Philippine J Microbiol Infect Dis 2008; 37(1): 20-32. 13.Haldar S, Sharma N, Gupta VK and Tyagi JS. Efficient diagnosis of tuberculous meningitis by detection of Mycobacterium tuberculosis DNA in cerebrospinal fluid filtrates using PCR. J Med Microbiol 2009; 58: 616–624. 15.Padilla E., Gonza´lez V, Manterola JM et al. Evaluation of two different cell lysis methods for releasing mycobacterial nucleic acids in the INNO-LiPA mycobacteria test. Diag Microbiol Infect Dis 2003; 46: 19–23. 2026 Ibrahim Medical College. All rights reserved.