<![CDATA[The Mycobacterium tuberculosis complex (MTBC) poses a significant health risk to both humans and animals because it can cause zoonotic tuberculosis (zTB). This disease can lead to substantial economic losses due to decreased productivity and increased mortality, particularly in livestock. The rpoB gene is a potential molecular marker for MTBC detection because it is highly conserved and specific. This study aimed to design specific primers targeting the rpoB gene and identify the optimal annealing temperature for conventional polymerase chain reaction (PCR) amplification. The rpoB gene sequence of M. tuberculosis H37Rv (accession number: NC_000962.3) was retrieved from the GenBank database of the National Center for Biotechnology Information (NCBI). Primers were designed using Clone Manager 9.0. The selected primers were tested using a temperature gradient in conventional PCR to determine the optimal annealing temperature. The amplification products were visualized by electrophoresis on a 1.5% agarose gel and confirmed by sequencing. The selected primers, SKRPOB_F148 5′-GTCCAGACCGATTCGTTCGAGTG-3′ and SKRPOB_R748 5′-CAGCGTCGATCGCATGATCTC-3′, successfully amplified an estimated 621-bp amplicon at an optimal annealing temperature of 57.5°C. The sequencing results showed 100% similarity between the primer-targeted sequence and the reference M. tuberculosis sequence. BLASTn analysis demonstrated 100% query coverage, 100% sequence identity, and an E-value of 0, confirming the high specificity of the primers. These findings indicate that the designed primers are specific and reliable for MTBC detection and have potential applications in molecular diagnostic methods.]]>
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