Background & aim of work:Tuberculosis(TB) is a critical contagious disease that primarily affects and represents asignificant public health problem all overthe world.
The ultimate diagnosis of Mycobacterium tuberculosis (MTB) is mainlyby microbiological tests including microscopy, culture and molecular methods.Both microscopy and culture have many drawbacks as low sensitivity for smearand lateness for culture. The initial step in the fast identification of TB bythe molecular method is DNA extraction. Methods: In this study, direct DNA extraction methodsof MBT from sputum samples were done by solid, digestion and phenol methods, while DNAextraction of MBT from culture isolateswere done by solid, boiling and Cetyl trimethylammonium bromide ( CTAB)methods. Results: Among 32positive sputum samples, the number of DNA extraction by phenol-chloroform-isoamyl alcohol method was21/32 (65.62%), followed by digestion method 14/32(43.75%) and the least onewas solid method 1/32(2.5%).
Thenumber of positive extracted DNA were significantly (P < 0.05) lower thanthe number of negative DNA extract obtained by solid method. Forculture 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 solidmethod. However, in CTAB method, the positive results didn't differsignificantly from the negative results. Conclusions: For DNA extraction from sputum, the bestmethod was phenol-chloroform-isoamyl alcohol method(mean rank 2.
34), while for extraction from culture isolates, the best methodwas boiling method (mean rank 2.45). The worst method of extraction for bothsputum and culture was solid method. Recovery of high yield DNA from MTB cultureis easier than sputum.
Key words: MTB, DNAextraction, PCR Introduction Tuberculosis(TB) is a critical infectious disease that primarily affects the lungs and ismore common in developing countries(Thwaites et al., 2002). In the21st century, it continues to be a significant problem for worldpublic health especially withthe development and rising of drug resistant TB (Abdelaalet al., 2009). The cornerstone of holding the spreadof TB includes quick diagnosis, proper case finding, immediate initiation ofeffective therapy and contact tracing to arrest further transmission.
Recentadvances in molecular biology methods have led to rapid identification of mycobacterialDNA (Abdelaal etal., 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 microscopymethod and the extended period of LJ culture lasting for many weeks. The evolution in molecular biology provides a molecularmethod that rapidly identifies MTB which is reflected on starting propertreatment early and holding its spread (Aldous et al., 2005).The efficacy of these methods relies on the type of sample, processing methodand the PCR steps.
The initial criticalstep in PCR is the DNA extraction from mycobacterial cells, yielding asufficient and pure DNA for effective PCR test (VanEmbden et al., 1993). Several molecular techniques for the diagnosisof TB are suggested and advocated by several studies (Mir et al., 2008). The basic interest of these studies isto establish a simple, precise and cheap technique (Takahashi et al., 2012).In addition, the convenient PCR depends on the selection of the most suitableextraction method and the target to be amplified (Adams et al., 2013).
There are many restricting factors facing the success of MTB DNAextraction techniques from clinical samples such as the slow generation timeresulting in a few number of organism, rigid cell wall rich in lipids that interferewith 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 andthe plenty of polysaccharides in the wall of mycobacteria. The positive aspectof polysaccharides is that it helps the isolation of intact bacteria from contaminatingmaterial(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 asheating to 100 ?C in a suitable buffer disrupts the bonds between the cell wall lipidsresulting in adequate DNA extraction. It is also a simple, easy and cheapmethod 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. Phenolis a strong proteolytic, corrosive and caustic agent that dissolves the cellwall of mycobacteria by its solubilizing and denaturing effects on both proteinsand lipids. In addition, the chloroform used in this method augment the effectof 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.
MethodsClinical Samples: 32 positive sputum and 40positive LJ culture specimens from the same tuberculous patients were collectedfrom Mansoura University Hospital, Clinical Pathology Department, from January2017 to June 2017. Each of sputum and culture specimens was subjected to DNAextraction by different methods. A) Methods of DNA extraction methodsfrom sputumPreparation of sputum for DNAextraction: Positive sputum samples were decontaminatedby mixing with equal volume of 4%NaOH for 30 min at 37 ?C with interruptedvortexing. Then samples were centrifuged at 6000 rpm and pellets were washedtwice with sterile distilled water by centrifugation. The pellet was suspendedin 1.
5ml of Tris-EDTA buffer (TE) to make homogenous suspension and finally wasequally divided into three sterile tubes for DNA extraction by different methods(WHO,1998). 1) Solidphase absorption Using QIAamp DNAMini Kit as mentioned by manufacturer’s instructions (QIAGEN, Hilden, Germany). 2)Digestion buffer method 2-3 ml of digestion buffer 500 mM Tris HClwith pH 9.0, 20 mM EDTA, 10 mM NaCl and 1%Sodium dodecyl sulfate (SDS) wasaseptically added to 0.
5 ml of the prepared pellet in a 10 ml tube, incubatedovernight at 60°C, and then vortexed for 20 seconds. 0.5 ml phenol was added to0.9 ml sample, vortexed for 20 seconds and centrifuged for 5 min at maximumspeed (14.
000 x g). The aqueous phase was transferred to a fresh tube, containing0.5 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 tubecontaining 35 ?l 3 M Na acetate and 800 ?l absolute ethanol, mixed andincubated for 20 min at -20°C and then centrifuged for 30 min at roomtemperature with maximum speed. The supernatant was discarded and the pellet waswashed with 500 ?l 70% ethanol and centrifuged for 5 min at maximum speed. The DNApellet was dried, re-suspended in 50-200 ?l 1x TE and stored at -20°C untilfurther use (Abbadi et al.,2001).
3)Phenol-chloroform-isoamyl alcoholmethod The pellet was re-suspended in 75 ?L of TE1X and 25 ?L of lysozyme solution (final concentration of 2.5mg/ml) was addedand incubated for 30 min at 37oC. 3.0 ?L of proteinase K (finalconcentration of 150 ?g/ml) and 20 ?L of10% SDS (final concentration of 1%) were added and completed with TE to a finalvolume 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 transferredto a clean microcentrifuge tube with the addition of 30 ?L of sodium acetate 3M with pH 4.
8. DNAwas precipitated with 1 volume isopropanol (300 ?L), agitated manually, andcentrifuged at 14,000 x g for 15 min. The supernatant was discarded and cold ethanol70% (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 TEbuffer (Yang et al.
, 2000).B) Methodsof DNA extraction from LJ cultureCulture on LJ:All collectedsamples were subjected to decontamination and concentration according to Khamiset al., 2004 and cultured on LJ solid medium,slopes were inspected weekly for up to 8 weeks and suspected growth wasconfirmed by ZN stain.1) Solid phase absorptionUsing QIAamp DNAMini Kit as mentioned by manufacturer’s instructions (QIAGEN, Hilden, Germany). 2) Boiling method: The simplest way of DNA release frommycobacterial suspension is boiling for 10 to 15 min in distilled water in cleanmicrocentrifuge tubes and kept at – 20 ° C until used (Abdelaal et al.,2014).
3)N- cetyl-N,N,N-Trimethylammonium bromide (CTAB) method: At least one loopfull of colonies wastransferred into a microcentrifuge tube containing 400 ul of 1xTE buffer, then heatedfor 20 min at 80°C to kill the cells, and cooled to room temperature. 50 ul of 10mg/ml lysozyme was added, vortexed and incubated at least 1 hour at 37°C. 75 ul10 % 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 prewarmingat 65°C,vortexed until the liquid content becomes white (“milky”) andincubated 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 temperaturefor 5 minutes at 12,000 g.
The aqueous supernatant was transferred to a freshmicrocentrifuge tube and 450ul isopropanol was added, incubated 10 min on iceand centrifuged 15 min at room temperature. The last step was to discard thesupernatant and wash the pellet with 1 ml of 70% ethanol and centrifuge(approximately 5 min at room temperature), then the pellet was dried and dissolvedin 100 ul of 1x TE buffer (Van Embden et al., 1993).
Purity of Extracted DNA The purity of the extracted DNA was checked bymeasuring the absorbance at 260 and 280nm. For pure DNA extract, the ratio ofA260/A280 should be between 1.8 and 2.0.
Conventional PCR amplification protocol: For themolecular identification by PCR, a pair of universally accepted primers wasused to amplify a fragment of the insertion sequence IS6110, a specificsequence for the MTB (26). For the PCR reaction, 24?L of 1xPCR-Master Mix (Fermentas™, USA), 3.0?L of each primer (INS-15′-CGTGAGGGCATCGAGGT GGC-3′ and INS-2 5’GCGTAGGCGTCGGTGACAAA-3′) and 6.
0?L ofgenomic 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 30minute, 55°C for 30 seconds and 72°C for 1 minute and a final cycle of 72°C for10 minutes. Subsequently, 5?L of amplified product was loaded on 1.5% agarosegel along with the molecularweight marker,separated by electrophoresis and stained with ethidium bromide (Azar and Abdulrazagh, 2007).