Development of Polymerase Chain Reaction–High-Resolution Melt Assay for Waterborne Pathogens <i>Legionella pneumophila</i>, <i>Vibrio parahaemolyticus</i>, and <i>Camplobacter jejuni</i>
2024
Shannon M. Carr | Kelly M. Elkins
<i>Legionella pneumophila</i> is the waterborne pathogen primarily responsible for causing both Pontiac Fever and Legionnaire’s Disease in humans. <i>L. pneumophila</i> is transmitted via aerosolized water droplets. The purpose of this study was to design and test primers to allow for rapid polymerase chain reaction (PCR) melt detection and identification of this infectious agent in cases of clinical or emergency response detection. New PCR primers were designed for this species of bacteria; the primer set was purchased from IDT and the target bacterial DNA was purchased from ATCC. The <i>L. pneumophila</i> primers targeted the macrophage infectivity potentiator gene (<i>mip</i>), which inhibits macrophage phagocytosis. The primers were tested for specificity, repeatability, and sensitivity using PCR–high-resolution melt (HRM) assays. The primer set was found to be specific to the designated bacteria and did not amplify the other twenty-one species from the panel. The <i>L. pneumophila</i> assay was able to be multiplexed. The duplex assay consists of primers for <i>L. pneumophila</i> and <i>Vibrio parahaemolyticus</i>, which are both waterborne pathogens. The triplex assay consists of primers for <i>L. pneumophila</i>, <i>V. parahaemolyticus</i>, and <i>Campylobacter jejuni</i>. The unique melting temperature for the <i>L. pneumophila</i> primer assay is 82.84 ± 0.19 °C, the <i>C. jejuni</i> assay is 78.10 ± 0.58 °C, and the <i>V. parahaemolyticus</i> assay is 86.74 ± 0.65 °C.
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