Design of an internal control (IC) for molecular diagnosis of Mycobacterium tuberculosis
Subject Areas : Molecular Microbiology
Mohammad Hassan Shahhosseini
1
,
Maryam Ghahri
2
,
Elham Moslemi
3
1 - Associated Professor, Department Microbiology,Shahr-e-Qods Branch, Islamic Azad University, Shahr-e-Qods, Iran.
2 - MS.c., Iranian Gene Fanavar Institute, Tehran, Iran.
3 - Assistant Professor, Department of Biology,Tehran Shargh Branch, Islamic Azad University, Tehran, Iran.
Keywords: PCR, Internal control, Mycobacterium tuberculosis. PCR-Cloning, Competitive,
Abstract :
Background & Objectives: Tuberculosis is the second reason of deaths caused by known infectious agents. Although molecular approaches, such as PCR, are suitable techniques for detection of Mycobacterium tuberculosis (MTB), these techniques suffer of variable results in different laboratory conditions. This study aimed to design, manufacture and apply an internal PCR control used for detection of MTB. Materials & Methods: To produce special MTB internal control, first individual PCR test primers was optimized for molecular detection of MTB, and then their sensitivity and specificity were measured. The composite primer for IC-MTB was also designed, replicated and colonized. The amplified IC-MTB was ligated to pTZ57R plasmid and was transformed into E. coli JM107. The minimum IC number in each PCR reaction was studied using dilution and PCR response spectrum with IC. Results: The size of MTB diagnostic products with individual primers and IC-MTB were was 245 bp and 660 bp, respectively, which is a suitable difference in the size aspect. Minimum IC number was identified 1000 for each reaction. Minimum and maximum sensitivity PCR test by IC for MTB DNA was determined 10 and 10 million bacteria, respectively. No unwanted products were observed in characteristic tests by different agents. Conclusion: Using an internal control as an inside control system we could detect the errors in MTB molecular diagnosis test. In fact, IC amplification is representative of correct procedure in amplification and detection steps.
