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Magnetically modified bacteriophage-triggered ATP release activated EXPAR-CRISPR/Cas14a system for visual detection
Juan Yao1, Zhang Zhang2, Hua Pei3
1NHC Key Laboratory of Tropical Disease Control, School of Tropical Medicine, Hainan Medical University, Haikou, Hainan, 571199, PR China; Nanobiosensing and Microfluidic Point-of-Care Testing, Key Laboratory of Luzhou, Department of Clinical Laboratory, The Affiliated Traditional Chinese Medicine Hospital, Southwest Medical University, Luzhou, Sichuan, 646000, PR China.
Abstract:
Burkholderia pseudomallei, widely distributed in tropical and subtropical ecosystems, is capable of causing the fatal zoonotic disease melioidosis and exhibiting a global trend of dissemination. Rapid and sensitive detection of B. pseudomallei is essential for environmental monitoring as well as infection control. Here, we developed an innovative biosensor for quantitatively detecting B. pseudomallei relies on ATP released triggered by bacteriophage-induced bacteria lysis. The lytic bacteriophage vB_BpP_HN01, with high specificity, is employed alongside magnetic nanoparticles assembly to create a biological receptor, facilitating the capture and enrichment of viable target bacteria. Following a brief extraction and incubation process, the captured target undergoes rapid lysis to release contents including ATP. The EXPAR-CRISPR cascade reaction provides an efficient signal transduction and dual amplification module that allowing the generated ATP to guide the signal output as an activator, ultimately converting the target bacterial amount into a detectable fluorescence signal. The proposed bacteriophage affinity strategy exhibited superior performance for B. pseudomallei detection with a dynamic range from 10^2 to 10^7 CFU mL-1, and a LOD of 45 CFU mL-1 within 80 min. Moreover, with the output signal compatible across various monitoring methods, this work offers a robust assurance for rapid diagnosis and on-site environmental monitoring of B. pseudomallei.
Insights
A novel biosensor detects Burkholderia pseudomallei using bacteriophage lysis and ATP release. This method offers rapid, sensitive detection crucial for controlling melioidosis and environmental monitoring.
Area of Science:
- Microbiology
- Biosensor Technology
- Molecular Diagnostics
Background:
- Burkholderia pseudomallei causes melioidosis, a fatal zoonotic disease with increasing global spread.
- Rapid and sensitive detection methods for B. pseudomallei are critical for environmental surveillance and infection control.
Purpose of the Study:
- To develop an innovative biosensor for the quantitative detection of viable Burkholderia pseudomallei.
- To utilize bacteriophage-induced lysis and ATP release for signal generation.
Main Methods:
- A lytic bacteriophage (vB_BpP_HN01) and magnetic nanoparticles were used for specific capture and enrichment of B. pseudomallei.
- Adenosine triphosphate (ATP) released post-lysis was detected using an EXPAR-CRISPR cascade reaction for signal amplification.
- Fluorescence signal output was correlated with bacterial concentration.
Main Results:
- The biosensor demonstrated a dynamic detection range from 10^2 to 10^7 CFU mL⁻¹.
- A limit of detection (LOD) of 45 CFU mL⁻¹ was achieved within 80 minutes.
- The system showed superior performance for B. pseudomallei detection.
Conclusions:
- The developed bacteriophage affinity biosensor provides a robust platform for rapid diagnosis of B. pseudomallei.
- The method is suitable for on-site environmental monitoring and infection control applications.
- The signal output compatibility enhances its versatility across different monitoring platforms.
