An Optimization of Precision-based Diagnostic Using atpE Genes Targeted Primers of a Drug Resistance Marker in Mycobacterium tuberculosis

An accurate and standardized diagnosis is needed to detect microbial resistance and changes in an organism's genome. The main problems faced in detecting Mycobacterium tuberculosis from clinical samples are the quality of the primers and the specificity of a gene target. This study aims to determine the main characteristics when evaluating primers’ s fitness generated from in silico experiments using Thermo Fisher Scientific® software and optimization efforts to increase sensitivity to clinical samples, this study also objectively evaluates the potential of atpE as a detection target for MTB using sputum specimens from a clinical setting. A total of ۲۵ clinical samples from pulmonary tuberculosis patients were extracted using the Zymo DNA Extraction® kit and ۱۱ of them were also extracted using the boiling method. DNA isolates were tested for concentration and purity with Nanodrops®. The primer was designed using the open-access software Thermo Fisher Scientific Oligo Primer design tool ®. Reference primer of atpE was obtained from previous research in vitro. Primary validation was carried out using gel electrophoresis. The ROC curve analysis was carried out with GraphPad Prism version ۸.۴. The atpE primers designed with Thermo Fisher Scientific® showed a ۱۰۰% detection rate against the positive control bacterial DNA of Mycobacterium tuberculosis H۳۷Rv. The overall atpE primers from Thermo Fisher Scientific® had a sensitivity of ۶۱.۵۴% and a specificity of ۱۰۰% compared to the reference primers against the clinical sample. In summary, the atpE gene is a good candidate for both species and drug-resistance genes targeted for PCR and genotype profiling in M. tuberculosis.

Keywords:

Molecular diagnostic , PCR-primer , Optimization , atpE , Mycobacterium Tuberculosis