Comparison of Different DNA Extraction Methods for the Diagnostic of Mycobacterium tuberculosis using Real-Time PCR Technique

During the study period (July to December 2016) a total of 188 samples (sputum and blood from each person) have been tested in the National Reference Laboratory (NRL) at the Institute of Chest and Respiratory Diseases / Baghdad. The calculation of sensitivity, specificity, positive predictive value and negative predictive value for any procedure used for detection will depend on the result of the standard procedure which is culture method, so we consider real positive case if it is positive in culture method and the same situation for the negative case. Three methods were submitted for DNA extraction, two from sputum and another one from blood. A Realline Kit and MTB Robogene kit successfully extracted DNA from Mycobacterium within all sputum samples. MTB Blood kit successfully extracted DNA from Mycobacterium within all blood samples A few general instructions about DNA extraction from microorganisms can be made. The range of DNA concentrations using Realline Kit, MTB Robogene kit and MTB blood kit were ( 1.65-14.43) ng/l, ( 1.34- 13.23) ng/l and ( 1.2-11.76) ng/l respectively. The mean concentration for the previously three mentioned kit were 9.7 ng/l, 7.3 ng/l and 3.2 ng/l. Detection of M. tuberculosis by real time PCR using Realline kit showed that among the total 188 specimens (Suspected Group), 112 (59.57 %) of the specimens were positive and 76 (40.4 %) negative which detected by Real-Time PCR using Realline Kit. All these samples are sputum. This study showed the percentage of sensitivity, specificity, PPV and NPV of Realline kit were 92.43 %, 97.1 %, 98.21 % and 88.15 % respectively. Detection of M. tuberculosis by real time PCR using MTB Robogene kit showed that among the total 188 specimens (Suspected Group), 111 (59.04 %) of the specimens were positive, and 77 (40.95 %) negative specimens by using MTB Robogene kit, the samples are sputum. the sensitivity, specificity, PPV and NPV of our study were 87.3 %, 89.85 %, 93.69 % and 80.51 % respectively. Detection of M. tuberculosis by real time PCR using MTB- DNA Blood showed that among total 188 specimens (Suspected Group), 59 (31.38 %) were positive in PCR and 129 (68.61 %) were negative, DNA was extracted directly from blood and then tested in MTB Robogene kit. The present showed the percentage of the sensitivity, specificity, PPV and NPV to be 47.89 %, 97.1 %, 96.6 % and 51.93.