Development and validation of a LAMP assay for detection of genetically diverse Lactococcus c2-type bacteriophages in...

Bacteriophages infecting Lactococcus spp. are a major cause of fermentation failures in the dairy industry. Among them, c2-type phages remain insufficiently studied, and rapid detection methods are limited. This study aimed to develop a loop-mediated isothermal amplification (LAMP) assay for sensitive detection of genetically diverse c2 phages. As part of this work, 12 prevalent c2 phages isolated from Russian dairy plants and 22 additional c2 phages were sequenced and analyzed. Conserved genomic regions were identified, and two primer sets were designed to account for the high genetic variability of c2 phages. Together, these primer sets enabled detection of all 34 tested c2 isolates despite varying numbers of mismatches within primer-binding regions. The assay detected 34 c2 isolates with varying numbers of mismatches in primer-binding regions. The detection limit was 10² copies/μL for plasmid DNA and 10²-10³ PFU/mL for bacterial lysates. Validation using industrial cheese whey samples confirmed assay applicability. Addition of high-fidelity Tersus polymerase improved mismatch tolerance, and in silico analysis predicted potential detection of up to 98% of publicly available c2 phages genomes. The developed assay provides a rapid and cost-effective tool for phage monitoring in dairy fermentations and may help reduce the risk of fermentation failures. Furthermore, the proposed approach may serve as a framework for developing diagnostic assays targeting other genetically diverse bacteriophage groups.