Discussion: didn’t differ significantly from the negative results. As

Discussion:

 

  

Continuous efforts are directed to
evolve a rapid, simple, precise and cheap method to extract mycobacterial DNA,
however, there is no established reference method. So, most of the laboratories
have developed their own extraction procedures after a prolonged time of  evaluation of different methods (Sambrook and Russell, 2001).

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Molecular methods as PCR enables quick
detection of MTB which is substantial to start treatment early and hold its
spread .The initial step in PCR is DNA extraction (Notle et al., 1993).The
mycobacterial DNA extraction techniques are very difficult due to the presence
of complex cell wall rich in lipids and the intracellular presence of pathogen
(Zumarraga
et al., 2005).

 

  

   In this study, direct DNA extraction methods
of 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) lower than the number of negative DNA extract obtained by solid method. However, for phenol method, the positive results didn't differ significantly from the negative results. As a consequence, the best method for DNA extraction from sputum in the current study was phenol-chloroform-isoamyl alcohol method (mean rank 2.34) as shown in table 1 and 3.    The phenol-chloroformthiocyanate guanidine for mycobacterial DNA extraction was superior over other methods owing to the good solubilizing and denaturing effect of both phenol and chloroform, helping the lysis of cell envelope and the removal of the lipid and protein layers of the mycobacterial cell wall (Palomo et al., 2017). The other characteristic of this method is the adjuvant effect of the use of SDS for 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 the multiple steps (Javadi et al.,2014).      After extraction, DNA purification is an essential step for the success of PCR by excluding inhibitors from the sputum samples. In order to achieve this, cell lysis was done by both physical and chemical steps with the addition of proteinase K and lysozyme. Also, ethanol was used for precipitation of extracted DNA and removal of organic contaminants. Solid phase absorption method is not a suitable routine method  for DNA extraction from sputum samples (Ruqaya  et al., 2014).    In the present study, DNA extraction methods of MTB from culture isolates were done by solid, boiling and CTAB methods. Among 40 positive cultures, 28(70%), 18 (45%) and 2 (5%)  DNA extracts were obtained by boiling, CTAB and solid methods respectively. The number of positive extracted DNA were significantly (P < 0.05) lower than the number of negative DNA extract obtained by solid method, However, the number of positive extracted DNA were significantly (P < 0.05) higher than the number of negative DNA extract obtained by  boiling method.  For CTAB method, the positive results didn't differ significantly from the negative results. As a result, the best method for extraction DNA from culture isolates was boiling method (mean rank 2.45) as shown in table 2 and 3.     Although, boiling is an easy, simple and cheap method for DNA extraction,  purification and precipitation 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 clinical specimens (Ruqaya et al., 2014).         Also, Cao et al., 2003 demonstrated that both the boiling and the phenolchloroform methods are good methods for 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 showed a poor A260/A280 ratio and weak band on agarose denoting the presence of DNA contaminants, while CTAB method yielded pure DNA. This variance could be attributed to the difference in samples and methodology between this study and the current one (Castro  et al., 2009).   So, by taking into consideration, the multiple characteristics of boiling methods, it could be used routinely in developing countries especially with a large number of samples to extract DNA from culture isolates.      The amplification of extracted DNA was done by using IS6110 primer. The insertion gene IS6110 has multiple copies which make the reaction more sensitive (Wildner  et al., 2011). The IS6110 target sequence is a recurrent repetition of 1,350 base pairs present in MTB complex species with different numbers of copies integrated into various chromosomal sites (Goldsborough and Bates, 201424). There are few reports stating that the IS6110 is absent in some MTB which is responsible for false negative (Sattar et al., 2014).  

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