In Silico and In Vitro Analyses of Previously Reported Primer Pairs for the Detection of Mycobacterium tuberculosis and Their Comparison With Two Novel Designed Primer Pairs

Background: The accurate and timely diagnosis of tuberculosis (TB) is crucial in the effective management and control of this infectious disease. Several molecular tests have been developed and are currently utilized for the diagnosis of Mycobacterium tuberculosis (MTB) in suspected patients. This study aimed to investigate various criteria affecting both the analytical and clinical specificity and sensitivity of primer pairs derived from published literature. The study also compared these established primer pairs with two novel primer pairs using in silico and in vitro methodologies. Methods: All studies related to the detection of MTB using polymerase chain reaction (PCR) and quantitative PCR (qPCR) were included based on established criteria. Eventually, all reported primer pairs were analyzed using in silico online software tools. Results: Among the 386 primarily retrieved articles, 98 met the eligibility criteria and were included for data extraction and analysis. Of these, 85 studies (86.7%) employed endpoint PCR, while 13 studies (13.3%) utilized q-PCR. All reported primers were thoroughly analyzed, resulting in the selection of six primers. Approximately 50% of the analyzed primer pairs targeted IS6110 and demonstrated a clinical sensitivity exceeding 80%. The rpoB gene revealed that nearly 35% of the primer pairs exhibited a clinical sensitivity greater than 80%. Conclusion: The newly designed primers, based on in silico analysis, represented favorable clinical sensitivity and specificity. Consequently, PCR using these primers may prove to be highly beneficial for the diagnosis and management of TB in low-resource settings.