MULTIPLEX PCR FOR SP-D rs3088308 AND CD36 rs1761667 GENE POLYMORPHISMS IN ACTIVE TUBERCULOSIS AND LATENT TUBERCULOSIS PATIENTS

Tuberculosis (TB) remains a leading cause of morbidity and mortality worldwide, particularly in developing countries such as Pakistan. Despite notable progress in diagnostic strategies, a large proportion of individuals with active tuberculosis (ATB) and latent tuberculosis infection (LTBI) remain undiagnosed, primarily due to limitations in diagnostic accessibility and sensitivity in resource-constrained settings. Genetic variations play a crucial role in determining host susceptibility to TB. Among these, polymorphisms in Surfactant Protein-D (SP-D) rs3088308 and Cluster of Differentiation 36 (CD36) rs1761667 have been associated with differential immune responses in ATB and LTBI. This study was planned to develop and optimize a multiplex Tetra-Primer Amplification Refractory Mutation System PCR (Tetra-ARMS PCR) assay for the simultaneous genotyping of these two polymorphisms in a single reaction; however, the optimization was unsuccessful. We assume the optimization procedure was challenged by factors such as substantial differences in primer annealing temperatures, minimal variation in amplicon sizes, the presence of multiple primers (eight in total), and primer-dimer formation. These constraints limited the assays reproducibility and efficiency. To improve multiplexing performance, primer redesign and re-optimization of PCR parameters are recommended. Furthermore, alternative molecular techniques, including high-resolution melting (HRM) analysis or allele-specific PCR, may provide more robust and reliable detection of these polymorphisms within diverse MTB infected populations.