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Target recognition initiated reverse hybridization mediated cascade amplification for sensitive Pseudomonas
1Department of Pediatrics, Affiliated Hospital of Shaoxing University, No 999 Zhongxing South Road, Yuecheng District, Shaoxing City, 312000, Zhejiang Province, China.
Abstract:
Pseudomonas aeruginosa (P. aeruginosa) is a significant opportunistic pathogen associated with nosocomial infections, particularly in pediatric populations, where it can lead to severe clinical manifestations, including P. aeruginosa-associated meningitis. To meet the critical need for highly sensitive detection of P. aeruginosa, a novel fluorescent biosensor utilizing a three-way junction (TWJ) probe has been developed. This biosensor capitalizes on the specific binding interaction between P. aeruginosa and a tailored aptamer embedded within a DNA TWJ structure. Upon target binding, the double-stranded DNA branches of the TWJ undergo a conformational rearrangement, resulting in the formation of two distinct DNA "Y" junction structures. These structures are subsequently linked by a designed sequence, initiating a DNA polymerase/endonuclease-mediated strand displacement amplification process. The TWJ-based biosensor offers several key advantages: (i) the integration of the aptamer sequence within the TWJ probe ensures high specificity for target recognition, and (ii) the subsequent enzymatic amplification significantly enhances the sensitivity of detection. Under optimized experimental conditions, the biosensor demonstrated a broad linear detection range from 10 to 10 to 105 cfu/mL, with an exceptionally low limit of detection of 4.12 cfu/mL. Recovery studies further confirmed the reliability and robustness, highlighting its potential for clinical implementation. This innovative bio-sensing strategy represents a significant advancement in diagnostic technology, offering a promising tool for the early and accurate detection of infectious diseases in pediatric patients, with potential applications in improving clinical outcomes.
Insights
A new fluorescent biosensor detects Pseudomonas aeruginosa (P. aeruginosa) with high sensitivity using a DNA three-way junction probe. This advancement offers rapid and accurate diagnosis for pediatric infections.
Area of Science:
- Biotechnology
- Molecular Diagnostics
- Infectious Disease Detection
Background:
- Pseudomonas aeruginosa (P. aeruginosa) is a major opportunistic pathogen causing nosocomial infections, especially in children.
- P. aeruginosa-associated meningitis presents severe clinical challenges in pediatric populations.
- Accurate and sensitive detection methods for P. aeruginosa are crucial for timely intervention.
Purpose of the Study:
- To develop a novel fluorescent biosensor for highly sensitive detection of P. aeruginosa.
- To leverage a DNA three-way junction (TWJ) probe integrated with an aptamer for specific target recognition.
- To enhance detection sensitivity through enzymatic amplification for improved clinical diagnostics.
Main Methods:
- Development of a fluorescent biosensor incorporating a DNA TWJ probe with a P. aeruginosa-specific aptamer.
- Utilizing aptamer binding to induce conformational changes in the TWJ structure.
- Employing DNA polymerase/endonuclease-mediated strand displacement amplification for signal enhancement.
Main Results:
- The biosensor demonstrated high specificity for P. aeruginosa detection.
- Achieved a broad linear detection range from 10 to 105 cfu/mL.
- Established an exceptionally low limit of detection of 4.12 cfu/mL.
- Recovery studies confirmed the biosensor's reliability and robustness.
Conclusions:
- The developed TWJ-based biosensor offers a sensitive and specific method for P. aeruginosa detection.
- This innovative bio-sensing strategy shows significant potential for early and accurate diagnosis of pediatric infectious diseases.
- The technology could improve clinical outcomes by enabling prompt identification of P. aeruginosa infections.
