Discussion: Continuous efforts are directed toevolve a rapid, simple, precise and cheap method to extract mycobacterial DNA,however, there is no established reference method.
So, most of the laboratorieshave developed their own extraction procedures after a prolonged time of evaluation of different methods (Sambrook and Russell, 2001). Molecular methods as PCR enables quickdetection of MTB which is substantial to start treatment early and hold itsspread .The initial step in PCR is DNA extraction (Notle et al., 1993).Themycobacterial DNA extraction techniques are very difficult due to the presenceof complex cell wall rich in lipids and the intracellular presence of pathogen(Zumarragaet al.
, 2005). In this study, direct DNA extraction methodsof MTB from sputum samples were done by solid, digestion and phenol methods. Among 32 positive sputum samples, 21 (65.62%) , 14(43.75%)and 1(2.
5%). The DNA extracts were obtained by phenol-chloroform-isoamyl alcohol method,digestion method and solid method respectively.The number of positive extracted DNA were significantly (P < 0.05) lowerthan the number of negative DNA extract obtained by solid method.However, for phenol method, the positive results didn't differ significantlyfrom the negative results.
As aconsequence, the best method forDNA extraction from sputum in thecurrent study was phenol-chloroform-isoamyl alcohol method(mean rank 2.34) as shown intable 1 and 3. The phenol-chloroformthiocyanate guanidine for mycobacterial DNA extractionwas superior over other methods owing to the good solubilizing and denaturingeffect of both phenol and chloroform, helping the lysis of cell envelope and theremoval of the lipid and protein layers of the mycobacterial cell wall (Palomo et al.
, 2017). Theother characteristic of this method is the adjuvant effect of the use of SDSfor cell wall lysis and proteinase K for dissolving proteins. On the other side,the drawbacks of this method included the toxicity of phenol-chloroform and themultiple steps (Javadi et al.,2014). After extraction, DNA purification is an essential step for the successof PCR by excluding inhibitors from the sputum samples.
In order to achievethis, cell lysis was done by both physical and chemical steps with the additionof proteinase K and lysozyme. Also, ethanol was used for precipitation ofextracted DNA and removal of organic contaminants. Solid phase absorptionmethod is not a suitable routine method for DNA extraction from sputum samples (Ruqaya et al., 2014). In thepresent study, DNA extraction methods of MTB from culture isolates were done bysolid, boiling and CTAB methods.
Among40 positive cultures, 28(70%), 18 (45%) and 2 (5%) DNA extracts were obtained by boiling, CTABand solid methods respectively. The number of positive extractedDNA were significantly (P < 0.05) lower than the number ofnegative DNA extract obtainedby solid method, However, the number of positive extractedDNA were significantly (P < 0.05) higher than the number ofnegative DNA extract obtainedby boiling method.
For CTAB method, the positive results didn’tdiffer significantly from the negative results. As a result, the best methodfor extraction DNA from culture isolates was boiling method (mean rank 2.45) asshown in table 2 and 3. Although, boiling is an easy, simple and cheap method for DNAextraction, purification andprecipitation steps were not done leading to low amount and quality of DNA. So,this method is suitable to extract DNA from culture and not from clinicalspecimens (Ruqaya et al.
, 2014). Also, Cao et al., 2003demonstrated that both the boiling and the phenolchloroform methods are good methodsfor both DNA extraction and amplification.
On the other hand, Ruqaya et al., (2014)stated that Boiling methods yielded low quantity of DNA compared to CTAB method,while solid phase method didn’t yield any DNA. Moreover, boiling method showeda poor A260/A280 ratio and weak band on agarose denoting the presence of DNAcontaminants, while CTAB method yielded pure DNA. This variance could beattributed to the difference in samples and methodology between this study andthe current one (Castro et al., 2009).
So, by taking into consideration, themultiple characteristics of boiling methods, it could be used routinely indeveloping countries especially with a large number of samples to extract DNAfrom culture isolates. Theamplification of extracted DNA was done by using IS6110 primer. The insertiongene IS6110 has multiple copies which make the reaction more sensitive (Wildner et al., 2011). The IS6110target sequence is a recurrent repetition of 1,350 base pairs present in MTBcomplex species with different numbers of copies integrated into variouschromosomal sites (Goldsborough and Bates, 201424).There are few reports stating that the IS6110 is absent in some MTB which isresponsible for false negative (Sattaret al.