# Development of Polymerase Chain Reaction–High-Resolution Melt Assay for Waterborne Pathogens Legionella pneumophila, Vibrio parahaemolyticus, and Camplobacter jejuni

## TL;DR

This study developed a PCR-based method to quickly detect and identify three waterborne pathogens, Legionella pneumophila, Vibrio parahaemolyticus, and Campylobacter jejuni, using high-resolution melt analysis.

## Contribution

The novel contribution is the design of specific PCR primers enabling rapid and accurate detection of three waterborne pathogens in a multiplexed assay.

## Key findings

- The L. pneumophila primer set was specific and did not cross-react with 21 other species.
- The method was successfully multiplexed to detect L. pneumophila, V. parahaemolyticus, and C. jejuni simultaneously.
- Each pathogen had a distinct melting temperature, allowing for clear differentiation in HRM analysis.

## Abstract

Legionella pneumophila is the waterborne pathogen primarily responsible for causing both Pontiac Fever and Legionnaire’s Disease in humans. L. pneumophila 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 L. pneumophila primers targeted the macrophage infectivity potentiator gene (mip), 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 L. pneumophila assay was able to be multiplexed. The duplex assay consists of primers for L. pneumophila and Vibrio parahaemolyticus, which are both waterborne pathogens. The triplex assay consists of primers for L. pneumophila, V. parahaemolyticus, and Campylobacter jejuni. The unique melting temperature for the L. pneumophila primer assay is 82.84 ± 0.19 °C, the C. jejuni assay is 78.10 ± 0.58 °C, and the V. parahaemolyticus assay is 86.74 ± 0.65 °C.