The rapid and accurate detection of antibiotic-resistant bacteria is crucial for effective infection control and treatment. Current diagnostic methods often involve multiple steps, including bacterial culturing, identification, and antibiotic susceptibility testing, which can be time-consuming and resource-intensive. Isothermal amplification techniques offer a faster alternative, but the complexity of the reaction mixtures and the need for sample purification often limit their practical application in clinical settings. Endonucleases, particularly CRISPR/Cas systems, have shown potential for molecular diagnostics due to their sequence-specific catalytic activity. However, challenges remain, including stringent requirements for cleavage sites and the instability of dual RNA tools. This paper explores the use of Argonaute (Ago) proteins, which offer advantages such as precise cleavage without requiring specific sequence motifs and increased stability compared to RNA-based systems. While previous research has used Ago proteins in conjunction with amplification techniques, this study aims to develop a simpler, amplification-free method for detecting antibiotic-resistant bacteria.

Existing methods for detecting antibiotic-resistant bacteria often rely on techniques like PCR or LAMP, which require multiple steps, specialized equipment, and skilled personnel. While CRISPR-Cas systems offer promise for rapid and sensitive detection, they are not without limitations. The need for a specific protospacer adjacent motif (PAM) sequence restricts their target range, and the use of dual RNA guides can lead to cost and stability issues. Ago proteins, homologous to the eukaryotic Ago family, provide an attractive alternative. They cleave target nucleic acids precisely through base pairing with a guide DNA, without needing a PAM sequence. Several previous studies have explored the use of Ago proteins in diagnostic applications; however, these methods often involve additional enzymes and amplification steps, leading to complex procedures and potentially hindering their widespread use. This study aims to overcome these limitations by creating a simpler, one-step detection method using Ago proteins and an artificial nucleic acid circuit.

The researchers developed the ANCA (artificial nucleic acid circuit with Ago protein) method, a one-step, amplification-free, isothermal DNA detection system. ANCA exploits the programmable sequence-guided cleavage activity of Ago proteins. The method uses a rationally designed DNA complex with self-reporting capabilities, incorporating target recognition and cleavage activity of the Ago system into a positive feedback loop. This loop enables high specificity and exponential signal amplification. The system requires only a single protein, simplifying manufacturing and reducing costs compared to other nucleic acid detection circuits. The reaction is performed in a single step, and the target can be easily altered by modifying the nucleic acid recognition site. The ANCA assay leverages two guide DNAs (G1 and G2), a reporter (R) and its complement (R*), each with specific sequences designed for synergistic function. The reaction starts with the Ago protein forming complexes with G1 and G2, recognizing and cleaving target DNA, generating T1, which acts as another guide DNA. This process repeats, leading to signal amplification. The reporter (R) and its complement (R*) produce a fluorescent signal. The researchers optimized the ANCA method by adjusting reaction temperature, concentrations of various components (R*, Ago protein, Mg²⁺, NaCl, BSA), and testing different reporter designs to find optimal conditions for detection sensitivity. This optimized method was then used to detect KPC, IMP, VIM, NDM, and OXA-48 sequences, with limits of detection determined for each. The direct detection of antibiotic-resistant bacteria in human urine and blood samples, as well as from surface swabs using a 3D nanopillar array structure, was investigated. The ANCA method's diagnostic performance was evaluated using clinical samples (rectal swabs) from patients, comparing the results with PCR analysis. The sensitivity and specificity were calculated, and a receiver operating characteristic (ROC) analysis was performed. Finally, the method was tested in a portable isothermal nucleic acid amplification device.

The ANCA method demonstrated high sensitivity and specificity in detecting carbapenemase-producing Klebsiella pneumoniae (CPKP). The limits of detection (LOD) varied depending on the target sequence: 1.87 fM for KPC, 17.8 fM for IMP, 529 fM for VIM, 120 fM for NDM, and 144 fM for OXA-48. The ANCA method successfully detected CPKP directly from human urine and blood samples without DNA purification or amplification, achieving high detection rates (90-99%). Direct detection from surface swabs using a 3D nanopillar array further enhanced the method's applicability. In clinical samples (rectal swabs), the ANCA method showed excellent diagnostic performance, achieving 100% sensitivity and specificity for IMP and 100% sensitivity and 98.7% specificity for KPC, with an AUC of 0.999 for KPC and 1.0 for IMP. Using rectal swabs directly, without transport media, resulted in even higher sensitivity (100% for both KPC and IMP). The quantitative analysis comparing the amount of target DNA detected in samples with and without transport media indicated that using a rectal swab directly could provide more than 50 times higher sensitivity than using a swab immersed in transport media. The method's efficacy was also demonstrated using a portable isothermal device.

The ANCA method provides a significant advancement in the rapid and accurate detection of antibiotic-resistant bacteria. Its simplicity, one-step process, amplification-free nature, and high sensitivity and specificity address the limitations of existing methods. The successful direct detection from various sample types eliminates the need for complex sample preparation, making it suitable for point-of-care diagnostics. The integration with a 3D nanopillar array further enhances detection efficiency. The high correlation between ANCA results and PCR results from clinical samples validates the method's accuracy and reliability. This method's simplicity and high performance could facilitate timely interventions and reduce the spread of antibiotic-resistant infections.

The ANCA method represents a significant advancement in the rapid and accurate detection of antibiotic-resistant bacteria. Its unique combination of an artificial nucleic acid circuit and Ago protein-based cleavage offers a simple, one-step, amplification-free, and highly sensitive approach. The successful application to various sample types, including clinical specimens, demonstrates its potential for widespread use in point-of-care settings. Further optimization and validation in diverse clinical settings are warranted.

While the ANCA method showed remarkable performance, further optimization might be necessary to improve its sensitivity, particularly for less abundant target sequences. The study primarily focused on CPKP detection; additional validation with other antibiotic-resistant bacteria is needed to fully establish its broad applicability. The 3D nanopillar array swab is currently a prototype, and its scalability for large-scale clinical use needs to be evaluated.