Bioinformatics-Driven Systematic Molecular Typing and Rapid qPCR Detection of Escherichia coli Phages: Preliminary Validation with Isolates from Cattle Farms in Xinjiang

This study aimed to classify <i>Escherichia coli</i> phages using bioinformatics analysis systematically and to establish corresponding PCR and qPCR detection methods for rapid molecular typing and identification. Based on 419 complete <i>E. coli</i> phage genomes available in NCBI, phylogenetic and pan-genomic analyses were conducted to classify the phages at the family, subfamily, and genus levels and to identify highly conserved core genes. Specific primers targeting these core genes were designed, and their specificity, sensitivity, and reproducibility were verified using conventional PCR and dye-based qPCR. A total of 357 phages were successfully classified, encompassing 10 families, 20 subfamilies, and 67 genera. Pan-genomic analysis identified type-specific core genes within 16 taxa, including <i>Ackermannviridae</i> and <i>Demerecviridae</i>, for which 16 pairs of primers were designed. Validation using bacteriophages isolated from Xinjiang cattle farms showed distinct single PCR bands with high specificity, and the qPCR assay achieved a sensitivity of up to 10^-5 µg/µL. This study established an efficient and broad-spectrum molecular typing and detection method for <i>E. coli</i> phages, providing a powerful preliminary screening tool for phage selection.