Development of a Microfluidics-Based Quantitative Real-Time PCR to Rapidly Identify Photobacterium damselae subsp. damselae with Different Pathogenicity by Detecting the Presence of mcp or dly Gene

Abstract

As a marine bacterial pathogen, Photobacterium damselae subsp. damselae (PDD) is distributed in seawater worldwide. It can infect different animals as well as humans, even cause deaths. The highly conserved regions of PDD mcp gene on chromosome and dly gene on plasmid were selected as the target fragments to design the specific primers. Recombinant plasmid standard was prepared based on the primers. With GENECHECKER UF-150 qRT-PCR instrument as the platform, a fluorescence-based quantitative real-time PCR (qRT-PCR) method was established for the detection of PDD. This method can specifically detect PDD and distinguish the highly virulent strains. Additionally, the test results can be qualitatively judged by visualization, while the quantitative detection can be achieved through the standard curve calculation. The minimum limit of detection was 1.0×101 copies µL−1, and the detection time was less than 20 min. In summary, this new method has outstanding advantages, such as strong specificity, high sensitivity, and low site requirements. It is a rapid on-site detection technology for highly virulent PDD strains.