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Published on: March 30, 2015
Development of Polymerase Chain Reaction-High-Resolution Melt Assay for Waterborne Pathogens Legionella pneumophila,
Shannon M Carr1, Kelly M Elkins1
1Forensic Science Program, Chemistry Department, Towson University, 8000 York Road, Towson, MD 21252-0001, USA.
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.
Insights
New PCR primers rapidly detect Legionella pneumophila, a waterborne pathogen causing Legionnaires' disease. This specific assay enables quick identification in clinical and emergency settings.
Area of Science:
- Microbiology
- Molecular Biology
- Pathogen Detection
Background:
- Legionella pneumophila is a waterborne pathogen responsible for Pontiac Fever and Legionnaire's Disease.
- Transmission occurs through aerosolized water droplets, posing risks in various environments.
Purpose of the Study:
- To design and validate novel PCR primers for rapid detection and identification of L. pneumophila.
- To enable timely identification in clinical diagnostics and emergency response scenarios.
Main Methods:
- Designed and tested new PCR primers targeting the macrophage infectivity potentiator (mip) gene of L. pneumophila.
- Evaluated primer specificity, repeatability, and sensitivity using PCR-high-resolution melt (HRM) assays.
- Developed multiplex assays combining L. pneumophila detection with other waterborne pathogens like Vibrio parahaemolyticus and Campylobacter jejuni.
Main Results:
- The primer set demonstrated high specificity for L. pneumophila, with no cross-amplification observed in twenty-one other bacterial species.
- The assay was successfully multiplexed into duplex and triplex formats for simultaneous detection of multiple waterborne pathogens.
- Unique melting temperatures were established for L. pneumophila (82.84°C), C. jejuni (78.10°C), and V. parahaemolyticus (86.74°C).
Conclusions:
- The developed PCR-HRM assay provides a specific, sensitive, and rapid method for identifying L. pneumophila.
- Multiplexing capabilities enhance the assay's utility for detecting multiple waterborne pathogens concurrently.
- This tool is valuable for clinical diagnostics and public health emergency response to waterborne diseases.
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