Arnaud, Tia2025-07-082025-07-082025-04-282025-04-29http://hdl.handle.net/1993/39146Bacteriophage (phage) therapy is currently being explored as an alternative treatment strategy to combat infections caused by antibiotic-resistant bacteria. However, considerable emphasis has been placed on utilizing only strictly virulent phages for treatment whereas the use of temperate phages has been largely inadvisable due to their potential to transmit antibiotic resistance genes. Unfortunately, the current process of isolating and deducing the lifecycle of newly-isolated phages is often labour-intensive, costly, and time-consuming, highlighting the need for a more streamlined process. Therefore, the aim of this study was to develop a PCR-based assay to screen out temperate phages that infect Enterococcus faecalis and Staphylococcus aureus during high-throughput phage isolation. Using the integrase gene in temperate phages as a molecular marker, cluster analysis was performed to identify conserved sequences and design cluster-specific degenerate primers. Multiplex PCRs (mPCRs) were then optimized using a set of short DNA sequences representing the integrase genes, and tested on a panel of plaque picks from phages isolated against E. faecalis and S. aureus. mPCR results were compared with whole genome and amplicon sequencing to verify the presence integrase genes. Overall, this study revealed that specific primers can be designed to target integrase genes in a simple and rapid mPCR assay. However, host-encoded prophages were detected from contaminating host nucleic acid in plaque picks, resulting in false positives as indicated by the sequencing results. This method may still be adopted – albeit, with careful consideration – by assuming that any same-sized band amplified from both the plaque picks and bacterial hosts are the result of contamination, and are therefore negative results. Ultimately, the findings of this research showed that a PCR screening method that detects homologues present only in virulent phages may be a better alternative.engbacteriophagehigh-throughputtemperate phagephage isolationmolecular screeningintegrase