Real-Time Recombinase Polymerase Amplification (RPA) Detection of Pseudomonas aeruginosa Using Magnetic Nano-Beads for DNA Extraction

Abstract

Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous nonfermentative gram-negative bacillus, residing in nature widely as a conditional pathogen that is commonly isolated from nosocomial infection cases, having a larger genome (5.5–7 Mbp). P. aeruginosa possesses great environmental adaptability and higher mutation rates, which accounts for its ability to resist antibiotics. Furthermore, multi-antibiotics-resistant P. aeruginosa has recently been established to be responsible for increased nosocomial infection incidence. Therefore, to detect, diagnose, and treat this life-threatening infection early enough, we introduced a real-time recombinase polymerase amplification (RPA) method of rapid P. aeruginosa detection, which is time-saving, convenient, highly sensitive, and more specific than the traditional bacterial culture method. System effectiveness and efficiency were then determined by a series of experiments, where specific RPA primers and probes accurately distinguished P. aeruginosa from other common pathogenic bacteria with optimal RPA reaction procedures set at 39 °C for 20 min. Additionally, specific primers and probes were designed based on the oprF gene sequence of P. aeruginosa. Subsequently, a real-time RPA system was used to analyze 20 cases of different clinical samples for P. aeruginosa detection in blood, open wound swab, and sputum. Results indicated that this system was suitable for bacterial analysis of common clinical sample types. The DNA extraction method used in this study was based on the magnetic beads method. In conclusion, this study established that the real-time RPA system was a rapid, specific, and sensitive method for clinical P. aeruginosa detection, which can be integrated into automatic high-throughput detection machines.