A SYBR Green based multiplex Real-Time PCR assay for rapid detection and differentiation of ocular bacterial...

Ocular bacterial pathogenesis is a serious sight threatening infection due to several bacterial species like Staphylococcus aureus, Streptococcus pneumoniae and Pseudomonas aeruginosa which are predominant. It is necessary to expedite diagnosis of pathogens for early treatment. Hence, a SYBR Green based multiplex Real-Time PCR assay coupled with melting curve analysis has been developed for rapid detection and differentiation of Staphylococcus aureus, Streptococcus pneumoniae and Pseudomonas aeruginosa in a single reaction.

The assay was designed for simultaneous detection and differentiation of pathogens based on their distinct melting curve. The analytical specificity, sensitivity and reproducibility of the assay were examined using various reference strains. Clinical validation was carried out with 100 ocular samples collected from patients suffering from ocular infections.

Each reaction tested for the targets individually generated three non overlapping melting curves with well alienated peaks corresponding to each gene. Among 100 ocular samples tested, 40 samples diagnosed with positive results in RT-PCR. Thus assay showed 100% specificity with high sensitivity and reproducibility.

The developed assay consistently established as a rapid and accurate diagnosis of ocular bacterial pathogens compared to the conventional laboratory techniques. Such precise method would aid greatly in clinical management of devastating ocular infections.