CN-121992077-A - Method for rapidly and sensitively detecting pathogenic bacteria based on recognition-amplification space-time isolation strategy of M13 phage biological lever

Abstract

Bacterial infection detection is often limited by the sensitivity of traditional immunoassays, resulting from carrier diffusion limitations and signal load-recognition efficiency tradeoff due to steric hindrance. For this, we constructed an M13 phage-mediated "bioslevering" system that spatially separated recognition from signal amplification, pIII protein-displayed nanobodies to achieve target-specific capture, pVIII protein covalently linked to high density DNA probes as signal interfaces, triggering low leakage Catalytic Hairpin Assembly (CHA) -CRISPR/Cas13a cascade amplification (LUCAS), converting a single recognition event into multi-stage signal enhancement. The design effectively improves transduction efficiency and suppresses background interference. With Acinetobacter baumannii as a model, the system realizes a detection limit of 10 CFU/mL within 2 hours, and is stable in performance in the labeled bronchoalveolar lavage fluid, and has no cross reaction to common pathogens. This strategy provides a powerful support for sensitive and rapid detection of pathogens.

Inventors

• YANG YUJUN
• ZHANG CHENLU

Assignees

• 重庆医科大学国际体外诊断研究院

Dates

Publication Date

20260508

Application Date

20260212

Claims (10)

1. 1. A modular biosensing detection platform based on M13 phage, characterized by comprising: The M13 phage nano-scaffold utilizes an anisotropic structure thereof to realize the spatial decoupling of a target recognition function and a signal amplification function; A target recognition element, modified on the terminal pIII capsid protein of said M13 phage, for specific capture of a target analyte; A signal transduction element, highly densely modified on the side wall pVIII capsid protein of said M13 phage, for converting the recognition event into a nucleic acid signal.
2. 2. The detection platform of claim 1, wherein the target recognition element is a nanobody (Nanobodies) to a target analyte.
3. 3. The assay platform of claim 1, wherein the signal transduction element is a single-stranded DNA (ssDNA) of a specific sequence that is anchored to the pVIII protein surface by chemical coupling to form a high density signal transduction interface.
4. 4. The detection platform of claim 1, further comprising a secondary signal amplification module, wherein the secondary signal amplification module is a low Leakage Universal Cascade Amplification System (LUCAS) comprised of a low leakage catalytic hairpin self-assembly (CHA) reaction in cascade with a CRISPR/Cas signal amplification system.
5. 5. The assay platform of claim 4, wherein said low leakage catalytic hairpin self-assembly (CHA) is achieved by removing toehold fragments of hairpin 1 and hairpin 2 to different lengths.
6. 6. The CRISPR/Cas system is preferably a Cas13a system, which degrades the fluorescent reporter release signal by recognizing that the output product generated by the CHA reaction triggers trans-cleavage activity (trans-cleavage).
7. 7. Use of the detection platform according to any one of claims 1-5 for the preparation of a tool for detection of pathogens including, but not limited to, acinetobacter baumannii (Acinetobacter baumannii), staphylococcus aureus or other bacteria, viruses.
8. 8. A method for rapid pathogen detection based on the platform of claim 1, comprising the steps of: (1) Incubating the M13 phage modified with the target recognition element and the signal initiation element with a sample to be tested to form a target-phage complex; (2) Removing unbound phage by magnetic bead separation; (3) Introducing a secondary signal amplification component, and triggering cascade amplification reaction by a signal initiation element on the surface of the phage; (4) The change of the fluorescent signal is monitored, and the quantitative or qualitative analysis of the target object is realized.
9. 9. The method of claim 7, wherein the method has a limit of detection (LOD) of acinetobacter baumannii of 10 CFU/mL and a total process detection time of less than 2 hours.
10. 10. The method of claim 7, wherein the sample to be tested is a complex biological matrix comprising bronchoalveolar lavage fluid (BALF).

Description

Method for rapidly and sensitively detecting pathogenic bacteria based on recognition-amplification space-time isolation strategy of M13 phage biological lever Technical Field The invention belongs to the technical field of biosensing detection, and particularly relates to a modular detection platform based on the 'biological lever' effect of M13 phage, a construction method thereof and application thereof in rapid ultrasensitive detection of pathogens. Background Bacterial infections remain one of the major challenges threatening public health worldwide. Recent global disease burden studies have shown that about 770 thousands of deaths worldwide in 2019 are associated with bacterial infections, accounting for 13.6% of the total deaths in the current year. Of the 1400 pathogens known to humans, only about 20 pathogens cause the vast majority of clinical infections such as inflammatory bowel disease, diarrhea, diabetes, and pneumonia. Among them, with the continuous upgrade of antibacterial drug resistance (antimicrobial resistance, AMR), refractory infections caused by "ESKAPE" pathogens (including enterococcus faecalis, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, and enterobacter) are becoming a central threat to the global medical system. Because of the highly pathogenic and multi-drug resistant nature of these pathogens, infections are often accompanied by high mortality rates, treatment failure, and dramatic increases in medical costs. Therefore, to achieve effective prevention and control of bacterial infections and accurate treatment of infected patients, it is highly desirable to construct a rapid, highly sensitive and highly specific pathogen detection method. In clinical microbiology, bacterial culture is still the gold standard for pathogen identification due to its high accuracy and reliability. However, culture-based methods are time consuming and often require additional analytical techniques to identify pathogens. Immunoassays rely on antigen-antibody recognition to play a key role in clinical diagnosis and microbiological identification. These assays have high specificity and affinity and enable selective detection even in complex biological matrices. Most clinical microbiology laboratories still rely on post-culture immunoassays to identify pathogens, including immunoturbidimetry, latex agglutination, and enzyme-linked immunosorbent assays (ELISA). Although these conventional methods are widely used, they are often limited by limited signal amplification efficiency and significant background interference, which together result in low analytical sensitivity. In the prior art, in order to improve the signal intensity, large-size nano materials are generally adopted as signal carriers. However, this introduces an inherent tradeoff in that an increase in carrier size, while increasing signal loading, can result in both severe diffusion limitations and steric hindrance, thereby significantly reducing the capture efficiency of the recognition element for the target. This coupling of the "recognition and amplification" functions limits further improvement in immunoassay performance. Therefore, it is important to develop a detection strategy that can break this trade-off and achieve efficient signal conversion. M13 phage is a typical filamentous phage, densely packed and modifiable capsid protein that is highly regular and engineered into fine particle structures that can be used as functionalized nano-scaffolds. The PIII protein at the tail end of M13 can be used for presenting a specific ligand through phage display technology to complete target recognition, and the high-copy pVIII protein at the side wall is chemically functionalized to construct a signal amplification module. By virtue of the spatial functional isolation, M13 serving as a biological lever can obviously enhance signal output while guaranteeing identification accuracy, and realizes stable construction and efficient detection of an immunoassay system. Disclosure of Invention In order to solve the technical problems, the invention designs a space-time separation identification-amplification strategy. The core of the invention is to use the anisotropic filament structure of M13 phage as a "bio-lever" scaffold: 1. Space-time decoupling design, namely, a target recognition element (such as a nano antibody) is modified on pIII protein at the top end of phage, and a signal transduction element (such as a DNA probe) is modified on pVIII protein at the side wall at high density. This physical separation avoids interference of the signal carrier with the identification process. 2. The biological lever effect is that a weak initial recognition event (target is captured at the pIII end) can drive DNA probes on thousands of pVIII sites on the side wall to participate in subsequent reactions, so that the primary physical amplification of signals is realized. 3. LUCAS cascade System A low leakag