Rapid Detection Of Pseudomonas Aeruginosa Septicemia By Real-Time Fluorescence Loop-Mediated Isothermal Amplification Of Free Dna In Blood Samples

Background: Pseudomonas aeruginosa septicemia poses significant healthcare-related problems including high mortality rates and substantial resource utilization. Successful antibiotic treatment relies on the accurate and rapid identification of infectious agents. However, current traditional methods of bacterial identification require extended processing steps including bacterial overnight culture, biochemical reactions, and antibiotic susceptibility testing. The sequential steps associated with infection diagnosis lack the celerity and sensitivity that is required to facilitate efficient result determination. We describe a rapid, sensitive, loop-mediated isothermal amplification assay for the direct detection of Pseudomonas aeruginosa DNA from clinical patient plasma. This technique is referred to as the "LAMP-Pa" (loop-mediated isothermal amplification of Pseudomonas aeruginosa) assay. Methods: Three primer pairs were designed to amplify the oprl gene, which encodes a Pseudomonas aeruginosa outer membrane protein. LAMP-Pa was performed using a 25 mu l final volume at 65 degrees C. For comparative purposes, we also used a traditional method of bacterial identification to evaluate assay efficacy. Results: The procedure enables direct detection from clinical patient plasma within 20 minutes without the requirement for DNA purification. The detection limit pertaining to Pseudomonas aeruginosa identification is 2.8 ng of total DNA/mu l plasma. The diagnosis is specific to Pseudomonas aeruginosa, and the coincidence rate of LAMP-Pa and the traditional method was 100%. Conclusions: Our data indicate that the assay provides a sensitive, specific, and intuitive diagnostic tool for Pseudomonas aeruginosa septicemia detection.