The specifc DNA region of esxA gene for the target of PCR to determine Mycobacterium tuberculosis accurately

Abstract

The esxA gene is a potent virulence gene and also known as conserved gene potential as a target to diagnose Mycobacterium tuberculosis complex (MTBC) especially Mycobacterium tuberculosis (M. tuberculosis). The aim of this study was to evaluate the fidelity of specific DNA region of esxA gene as target for PCR to identify and determine species MTBC among Mycobacteria clinical isolates, compare with the culture method. Clinical isolate samples were collected randomly from January 2016 until July 2016 among Mycobacteria sputum isolates of suspected pulmonary tuberculosis (TB) patients in Dr. Soetomo Hospital, Surabaya, Indonesia. Samples were detected and identified using BACTEC MGIT 960 system (BD) culture method, then identified the MTBC TB Ag MPT 64 (SD Bioline). Furthermore, clinical isolates from culture method were amplified by using PCR based on esxA as gene target. Primer for esxA gene region was designed using Clone Manager 6, version 6.00. The amplicon was confirmed as positive result as DNA band appeared in 339bp. M. tuberculosis H37Rv strain was used as positive control, PCR mixture without DNA, and Mycobacterium fortuitum (M. fortuitum) isolates as negative control. Total 37 clinical isolates from sputum specimens which had been analyzed using PCR revealed that all clinical isolates were positive and in concordance with the result of standard culture method, MGIT. The conserved and specific DNA region of esxA gene with size of 339 bp for PCR have high accuracy for identification of M. tuberculosis that is important in determining diagnosis of MTBC infection.