Background & aim of work:
(TB) is a critical contagious disease that primarily affects and represents a
significant public health problem all over
the world. The ultimate diagnosis of Mycobacterium tuberculosis (MTB) is mainly
by microbiological tests including microscopy, culture and molecular methods.
Both microscopy and culture have many drawbacks as low sensitivity for smear
and lateness for culture. The initial step in the fast identification of TB by
the molecular method is DNA extraction.
Methods: In this study, direct DNA extraction methods
of MBT from sputum samples were done by solid, digestion and phenol methods, while DNA
extraction of MBT from culture isolates
were done by solid, boiling and Cetyl trimethylammonium bromide ( CTAB)
Results: Among 32
positive sputum samples, the number of DNA extraction by phenol-chloroform-isoamyl alcohol method was
21/32 (65.62%), followed by digestion method 14/32(43.75%) and the least one
was solid method 1/32(2.5%). The
number of positive extracted DNA were significantly (P < 0.05) lower than the number of negative DNA extract obtained by solid method. For culture isolates, the number of DNA extraction by boiling method was 28/40(70%), followed by CTAB method 18/40(45%) and the least one was solid method 2/40(5%). The number of positive extracted DNA were significantly (P < 0.05) lower than the number of negative DNA extract obtained by solid method. However, in CTAB method, the positive results didn't differ significantly from the negative results. Conclusions: For DNA extraction from sputum, the best method was phenol-chloroform-isoamyl alcohol method (mean rank 2.34), while for extraction from culture isolates, the best method was boiling method (mean rank 2.45). The worst method of extraction for both sputum and culture was solid method. Recovery of high yield DNA from MTB culture is easier than sputum. Key words: MTB, DNA extraction, PCR Introduction Tuberculosis (TB) is a critical infectious disease that primarily affects the lungs and is more common in developing countries (Thwaites et al., 2002). In the 21st century, it continues to be a significant problem for world public health especially with the development and rising of drug resistant TB (Abdelaal et al., 2009). The cornerstone of holding the spread of TB includes quick diagnosis, proper case finding, immediate initiation of effective therapy and contact tracing to arrest further transmission. Recent advances in molecular biology methods have led to rapid identification of mycobacterial DNA (Abdelaal et al., 2014). Microbiological methods are the clue for the laboratory diagnosis of TB. The lateness in TB diagnosis is due to the weak sensitivity of the microscopy method and the extended period of LJ culture lasting for many weeks. The evolution in molecular biology provides a molecular method that rapidly identifies MTB which is reflected on starting proper treatment early and holding its spread (Aldous et al., 2005). The efficacy of these methods relies on the type of sample, processing method and the PCR steps. The initial critical step in PCR is the DNA extraction from mycobacterial cells, yielding a sufficient and pure DNA for effective PCR test (VanEmbden et al., 1993). Several molecular techniques for the diagnosis of TB are suggested and advocated by several studies (Mir et al., 2008). The basic interest of these studies is to establish a simple, precise and cheap technique (Takahashi et al., 2012). In addition, the convenient PCR depends on the selection of the most suitable extraction method and the target to be amplified (Adams et al., 2013). There are many restricting factors facing the success of MTB DNA extraction techniques from clinical samples such as the slow generation time resulting in a few number of organism, rigid cell wall rich in lipids that interfere with cell wall lysis and finally the intracellular presence of the pathogen ( Zumarraga et al.,2005). Also, the purification of MTB DNA is hindered by the compound lipids and the plenty of polysaccharides in the wall of mycobacteria. The positive aspect of polysaccharides is that it helps the isolation of intact bacteria from contaminating material (Van Helden et al., 2001). Various physical and chemical methods are used for DNA extraction from MTB. Boiling is one of the physical methods used to extract mycobacterial DNA as heating to 100 ?C in a suitable buffer disrupts the bonds between the cell wall lipids resulting in adequate DNA extraction. It is also a simple, easy and cheap method for DNA extraction from culture, but not from clinical specimens (Ruqaya et al., 2014). On the other hand, phenol extraction is an example of chemical methods. Phenol is a strong proteolytic, corrosive and caustic agent that dissolves the cell wall of mycobacteria by its solubilizing and denaturing effects on both proteins and lipids. In addition, the chloroform used in this method augment the effect of phenol as it has the same action (Banavaliker, et al., 1998). The aim of work: The aim of this study was to compare DNA extraction of MTB from sputum and LJ culture isolates by different methods. Methods Clinical Samples: 32 positive sputum and 40 positive LJ culture specimens from the same tuberculous patients were collected from Mansoura University Hospital, Clinical Pathology Department, from January 2017 to June 2017. Each of sputum and culture specimens was subjected to DNA extraction by different methods. A) Methods of DNA extraction methods from sputum Preparation of sputum for DNA extraction: Positive sputum samples were decontaminated by mixing with equal volume of 4%NaOH for 30 min at 37 ?C with interrupted vortexing. Then samples were centrifuged at 6000 rpm and pellets were washed twice with sterile distilled water by centrifugation. The pellet was suspended in 1.5ml of Tris-EDTA buffer (TE) to make homogenous suspension and finally was equally divided into three sterile tubes for DNA extraction by different methods (WHO,1998). 1) Solid phase absorption Using QIAamp DNA Mini Kit as mentioned by manufacturer's instructions (QIAGEN, Hilden, Germany). 2) Digestion buffer method 2-3 ml of digestion buffer 500 mM Tris HCl with pH 9.0, 20 mM EDTA, 10 mM NaCl and 1%Sodium dodecyl sulfate (SDS) was aseptically added to 0.5 ml of the prepared pellet in a 10 ml tube, incubated overnight at 60°C, and then vortexed for 20 seconds. 0.5 ml phenol was added to 0.9 ml sample, vortexed for 20 seconds and centrifuged for 5 min at maximum speed (14.000 x g). The aqueous phase was transferred to a fresh tube, containing 0.5 ml phenol, vortexed for 20 sec and centrifuged for 5 min at maximum speed. Again the aqueous phase (approximately 350 ?l) was transferred to a fresh tube containing 35 ?l 3 M Na acetate and 800 ?l absolute ethanol, mixed and incubated for 20 min at -20°C and then centrifuged for 30 min at room temperature with maximum speed. The supernatant was discarded and the pellet was washed with 500 ?l 70% ethanol and centrifuged for 5 min at maximum speed. The DNA pellet was dried, re-suspended in 50-200 ?l 1x TE and stored at -20°C until further use (Abbadi et al., 2001). 3) Phenol-chloroform-isoamyl alcohol method The pellet was re-suspended in 75 ?L of TE 1X and 25 ?L of lysozyme solution (final concentration of 2.5mg/ml) was added and incubated for 30 min at 37oC. 3.0 ?L of proteinase K (final concentration of 150 ?g/ml) and 20 ?L of 10% SDS (final concentration of 1%) were added and completed with TE to a final volume of 200?L, incubated at 65 oC with occasional agitation. Extract DNA with 300 ?L of phenol/chloroform/isoamyl alcohol (25:24:1), centrifuged at 14,000 x g for 5 minutes, and the aqueous phase was transferred to a clean microcentrifuge tube with the addition of 30 ?L of sodium acetate 3M with pH 4.8. DNA was precipitated with 1 volume isopropanol (300 ?L), agitated manually, and centrifuged at 14,000 x g for 15 min. The supernatant was discarded and cold ethanol 70% (300 ?L) was added to the pellet and centrifuged at 14,000 x g for 5 min. The pellet was dried at room temperature and then re-suspended in 100 ?L TE buffer (Yang et al., 2000). B) Methods of DNA extraction from LJ culture Culture on LJ: All collected samples were subjected to decontamination and concentration according to Khamis et al., 2004 and cultured on LJ solid medium, slopes were inspected weekly for up to 8 weeks and suspected growth was confirmed by ZN stain. 1) Solid phase absorption Using QIAamp DNA Mini Kit as mentioned by manufacturer's instructions (QIAGEN, Hilden, Germany). 2) Boiling method: The simplest way of DNA release from mycobacterial suspension is boiling for 10 to 15 min in distilled water in clean microcentrifuge tubes and kept at – 20 ° C until used (Abdelaal et al.,2014). 3)N- cetyl-N,N,N-Trimethyl ammonium bromide (CTAB) method: At least one loopfull of colonies was transferred into a microcentrifuge tube containing 400 ul of 1xTE buffer, then heated for 20 min at 80°C to kill the cells, and cooled to room temperature. 50 ul of 10 mg/ml lysozyme was added, vortexed and incubated at least 1 hour at 37°C. 75 ul 10 % of SDS/proteinase K solution (5 M proteinase K, 10 mg/ml and 70 M 10% SDS) were added, vortexed shortly and incubated 10 min at 65°C. Then 100ul l5M NaCl and 100 ul CTAB/NaCl solution (4.1 g NaCl and 10 g CTAB in 100 M distilled water) were added after prewarming at 65°C,vortexed until the liquid content becomes white ("milky") and incubated for 10 min at 65°C. After that 750 ul of chloroform/isoamyl alcohol (24:1) was added, vortexed for10 seconds and centrifuged at room temperature for 5 minutes at 12,000 g. The aqueous supernatant was transferred to a fresh microcentrifuge tube and 450ul isopropanol was added, incubated 10 min on ice and centrifuged 15 min at room temperature. The last step was to discard the supernatant and wash the pellet with 1 ml of 70% ethanol and centrifuge (approximately 5 min at room temperature), then the pellet was dried and dissolved in 100 ul of 1x TE buffer (Van Embden et al., 1993). Purity of Extracted DNA The purity of the extracted DNA was checked by measuring the absorbance at 260 and 280nm. For pure DNA extract, the ratio of A260/A280 should be between 1.8 and 2.0. Conventional PCR amplification protocol: For the molecular identification by PCR, a pair of universally accepted primers was used to amplify a fragment of the insertion sequence IS6110, a specific sequence for the MTB (26). For the PCR reaction, 24?L of 1x PCR-Master Mix (Fermentas™, USA), 3.0?L of each primer (INS-1 5'-CGTGAGGGCATCGAGGT GGC-3' and INS-2 5'GCGTAGGCGTCGGTGACAAA-3') and 6.0?L of genomic DNA were mixed with 14.0?L of sterile nuclease free water. After this, the reaction was performed in the thermo cycler under the following conditions: initial cycle of 95ºC for 4 minutes, followed by 30 cycles of 95°C for 30 minute, 55°C for 30 seconds and 72°C for 1 minute and a final cycle of 72°C for 10 minutes. Subsequently, 5?L of amplified product was loaded on 1.5% agarose gel along with the molecular weight marker, separated by electrophoresis and stained with ethidium bromide (Azar and Abdulrazagh, 2007).