Establishment of loop-mediated isothermal amplification for rapid detection of Pseudomonas aeruginosa

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Pseudomonas aeruginosa is a highly pathogenic bacterium causing severe opportunistic infections, especially in immunocompromised individuals.
• Rapid and sensitive detection methods are crucial for timely treatment of P. aeruginosa infections, particularly in hospital settings like burn wards.

Purpose of the Study:

• To develop and validate a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of P. aeruginosa.
• To target the P. aeruginosa-specific gene hypothetical protein (GenBank ID: 882161) for diagnostic purposes.

Main Methods:

• Designed specific primers targeting the hypothetical protein gene of P. aeruginosa.
• Optimized reaction conditions for LAMP, including temperature, time, Mg2+ concentration, DNA polymerase, deoxynucleotide, and primer concentrations.
• Evaluated the method's specificity using 150 P. aeruginosa and 170 non-P. aeruginosa strains.
• Assessed sensitivity using plasmids with the target gene and spiked mouse blood, determining the detection limit.

Main Results:

• The optimized LAMP method demonstrated high specificity, correctly identifying all 150 P. aeruginosa strains and yielding negative results for all 170 non-P. aeruginosa strains.
• The sensitivity assay revealed a detection limit of 1 bacterium per reaction.
• Positive reactions were observed consistently for P. aeruginosa strains.

Conclusions:

• The developed LAMP method is a fast, specific, sensitive, and cost-effective tool for detecting Pseudomonas aeruginosa.
• This method is suitable for rapid identification of P. aeruginosa in clinical and potentially other relevant settings.