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Published on: June 25, 2020
Rapid On-Site Detection of Pseudomonas aeruginosa via ecfX-Targeted Loop-Mediated Isothermal Amplification
Xuliang He1, Meimei Zeng2, Wentao Bai3,4
1Department of General Surgery, The Affiliated Zhuzhou People's Hospital of Changsha Medical University, Zhuzhou 412000, China.
Abstract:
Pseudomonas aeruginosa (PA) is a significant pathogen of clinical concern that is frequently associated with multidrug resistance, leading to respiratory tract, wound, and hospital-acquired infections. To enable rapid and accurate detection, we developed a fluorescence-based loop-mediated isothermal amplification (LAMP) method, targeting the PA-specific ecfX gene. Among ten primer sets designed, the optimal set (EC2) was identified, and reaction conditions were optimized (Bst polymerase 320 U/mL, Mg2+ 8 mM, dNTP 1.4 mM, inner/outer primer ratio 1:8, 64 °C, 20 min). The assay demonstrated a detection limit that was comparable to a real-time polymerase chain reaction and immunochromatographic assays, but with a markedly reduced turnaround time. No cross-reactivity was observed with non-PA pathogens, and reproducibility tests confirmed high stability. In addition, the reliability of the results was further verified using 60 standard bacterial strains, and the feasibility of the assay was validated with 2 real soil samples and 1 water sample. This LAMP method offers a simple, rapid, and sensitive tool for on-site detection of PA, with potential applications in clinical diagnostics and public health surveillance.
Insights
A new fluorescence-based loop-mediated isothermal amplification (LAMP) method rapidly detects Pseudomonas aeruginosa (PA). This sensitive assay offers a faster alternative for clinical diagnostics and public health surveillance.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Pseudomonas aeruginosa (PA) is a multidrug-resistant pathogen causing serious infections.
- Accurate and rapid detection of PA is crucial for effective treatment and control.
Purpose of the Study:
- To develop a rapid, sensitive, and specific fluorescence-based loop-mediated isothermal amplification (LAMP) assay for detecting Pseudomonas aeruginosa (PA).
Main Methods:
- Designed and optimized ten primer sets targeting the PA-specific ecfX gene.
- Optimized reaction conditions including enzyme concentration, ion cofactors, nucleotides, primer ratios, temperature, and time.
- Evaluated assay sensitivity, specificity, reproducibility, and compared it with real-time PCR and immunochromatographic assays.
- Validated the assay using standard bacterial strains and environmental samples (soil and water).
Main Results:
- Identified an optimal primer set (EC2) and reaction conditions for the LAMP assay.
- Achieved a detection limit comparable to real-time PCR and immunochromatographic assays.
- Demonstrated no cross-reactivity with other pathogens and high reproducibility.
- Successfully validated the assay with clinical strains and environmental samples.
Conclusions:
- The developed fluorescence-based LAMP assay is a simple, rapid, and sensitive tool for on-site PA detection.
- This method has significant potential for clinical diagnostics and public health surveillance applications.
- Offers a valuable alternative to existing detection methods with reduced turnaround time.
