Construction of an internal amplification control for Mycobacterium tuberculosis polymerase chain reaction (PCR) test

Abstract

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB) which mostly affects the lungs. The disease causes deaths of many people every year. There are different methods to detect MTB such as skin test, staining, culture and molecular techniques. Polymerase chain reaction (PCR) is a simple and rapid method for the detection of MTB; however, positive and negative false results reduce the efficiency of this technique. The aim of this study was to design an internal amplification control (IAC) and apply it in MTB PCR test. PCR technique for MTB was optimized by using specific primers for IS6110 gene. The sensitivity and specificity of the test were determined. IAC was constructed with competitive strategy by PCR-cloning technique and the limitation range was determined. The PCR products of MTB and IAC were 245 and 660 bp, respectively on electrophoresis gel. The IC used in PCR testing of MTB is the competitive form in which the range was between 10 million and 10 bacteria and the most suitable internal control concentration for the mix was 1,000 plasmids. After making IC and using it in MTB amplification, it was observed that IC might guarantee the correctness of PCR reaction.

Keywords: Mycobacterium tuberculosis, polymerase chain reaction (PCR), internal control.

African Journal of Biotechnology Vol. 12(27), pp. 4277-4282