Rapid and sensitive qPCR-based quantification of Salmonella Gallinarum Bacteriophages cocktail (SAL3)

Abstract Bacteriophages have garnered significance as alternatives to antibiotics against multidrug-resistant pathogens. Their prophylactic and therapeutic use in poultry production is well documented against Salmonella spp. and E. coli . However, accurate and reliable quantification of each bacteriophage is crucial for cocktail development and quality assurance. However, conventional plaque assay method is laborious and requires at least 12 hours of incubation for phage quantification. Moreover, for a phage cocktail targeting a single host, plaque assay cannot differentiate between individual phages except when different phages produce morphologically different plaques. Hence, it can compromise the quality assurance during cocktail production. In this study, we developed a quantitative qPCR assay to identify and quantify three lytic bacteriophages (SGP9, SGP13, and Frank) within 'SAL3,' a bacteriophage cocktail formulated against Salmonella Gallinarum. Phage-specific primers and TaqMan probes were designed and optimized to achieve high sensitivity with a detection limit of 10 3 –10 4 phage genomes per mL (equivalent to 2–20 genomes per reaction). Standard curves generated from plasmids containing phage genome fragments showed excellent linearity, with a correlation coefficient (R 2 ) of 0.99. Comparative analysis of freshly prepared phage stocks using direct qPCR and the double-layer agar (DLA) method revealed a consistent, phage-dependent ratio between the two techniques. Direct qPCR detected approximately one log higher phage count than DLA and DNase treated qPCR assay, reflecting total phage genomes in the preparation rather than only infectious phages. Phage titer obtained after treating the phage prep with DNase I was comparable with DLA count. qPCR-based quantifications reduced processing time to under 3 hours with high reproducibility (CV < 5%) compared to the conventional DLA method, which requires over 12 hours. Multiplex qPCR assay enabled a rapid identification and quantification of individual phages in SAL3 cocktail. Additionally, DNase pretreatment reduced the contamination of free DNA both in the cocktail and lysate.