ANCA: artificial nucleic acid circuit with Argonaute protein for one-step isothermal detection of antibiotic-resistant bacteria

Carbapenemase-producing Enterobacteriaceae (CPE) pose a growing clinical threat, yet existing nucleic-acid-based diagnostics often require multistep processing, thermal cycling or complex isothermal chemistries that are incompatible with crude clinical samples. CRISPR/Cas12/13 diagnostics offer sensitive detection via collateral cleavage but depend on PAM/PFS constraints and can involve costly, less stable RNA guides. Argonaute (Ago) proteins provide programmable, sequence-guided nuclease activity without PAM requirements and can use DNA guides that are more stable and economical. The study aims to harness Ago’s cleavage properties within an artificial nucleic acid circuit (ANCA) to create a one-step, amplification-free, isothermal assay capable of directly detecting carbapenemase-producing Klebsiella pneumoniae (CPKP) from diverse sample types, thereby addressing the need for rapid, simple, and accurate point-of-care CPE screening.

The article surveys endonuclease-based diagnostics, highlighting CRISPR/Cas12 and Cas13 platforms that leverage collateral cleavage for signal amplification, while noting limitations such as PAM dependence and RNA guide instability/cost. It reviews Argonaute proteins’ roles in RNAi and programmable nucleic acid cleavage without PAM constraints, with the ability to use DNA guides. Prior Argonaute-based assays typically coupled Ago cleavage to upstream amplification (PCR, LAMP, ligase chain reaction) or exonuclease steps and often required multiple enzymes, increasing workflow complexity. Examples include RT-PCR followed by Ago-triggered reporter cleavage for SARS-CoV-2, LAMP-cDNA cleavage for respiratory viruses, PCR plus exonuclease I with Ago for foodborne pathogens, Ago-mediated pre-cleavage prior to amplification, and Ago integrated with ligation-based workflows. While these show promising specificity, amplification-free Ago assays previously exhibited relatively poor sensitivity, motivating development of a self-amplifying, Ago-driven circuit to simplify and sensitize detection.

Design and reaction principle: The ANCA method integrates Thermus thermophilus Argonaute (TtAgo) with a rationally designed DNA circuit comprising four oligonucleotide components: guide DNA 1 (G1), guide DNA 2 (G2), a reporter (R), and a reporter complement (R*). G1 and G2 are designed per NEB guidelines, and the system produces short trigger strands (T1, T2) that themselves act as guides, establishing a positive feedback loop. Ago/G1 and Ago/G2 bind the target DNA and cleave between the 10th and 11th nucleotides from the guide 5′ end, generating T1. Ago/T1 then cleaves R* to release an output and T2; Ago/T2 further cleaves R*, regenerating T1 and completing the autocatalytic cycle. Fluorophore/quencher-labeled reporters yield a fluorescence readout upon cleavage. Optimization: Reaction parameters were optimized for performance: 75°C reaction temperature; R* at 500 nM; Ago protein at 200 nM; MgCl2 at 10 mM; NaCl at 75 mM; BSA at 10 µg/mL. Reporter labeling was tuned to minimize steric hindrance (optimal when F/Q attached to R or R* sequence-only rather than both). Fluorescence was monitored at 1-min intervals using a Bio-Rad CFX Opus 96 RT-PCR system. Assay evaluation: Component omission controls (no target, no Ago, no G1/G2) showed negligible background. PAGE confirmed R* integrity in negative conditions and cleavage following complete ANCA reactions. Without R* (no feedback), only initial target cleavage and R processing occurred with no amplification, confirming the necessity of R* for exponential signaling. Analytical sensitivity: For KPC targets, threshold time (time to fluorescence=10,000 a.u.) correlated linearly with log concentration (R²=0.99) over 10 fM–10 nM; LOD was reported as 1.87 fM. For IMP, linearity spanned 1 fM–1 nM with an LOD of 17.8 fM (R²=0.99). Circuits for VIM, NDM, and OXA-48 also showed linearity over 1 fM–1 nM, with reported LODs of 529 nM (VIM), 120 nM (NDM), and 144 nM (OXA-48). The method demonstrated adaptability to multiple resistance gene targets including MRSA-associated sequences. Direct bacterial detection (no extraction/amplification): Cultured K. pneumoniae strains producing KPC or IMP were added directly to ANCA mixtures and incubated at 75°C. KPC circuits specifically detected K. pneumoniae (KPC) over wild-type and IMP strains; IMP circuits specifically detected IMP producers. Additional circuits for VIM, NDM, and OXA-48 discriminated resistant from wild-type strains. Clinical matrices: Human urine and blood were spiked with target bacteria. Autofluorescence was assessed and found minimal in the FAM channel. The KPC circuit detected K. pneumoniae in up to 99% of spiked urine and 90% of spiked blood samples; the IMP circuit detected P. aeruginosa (IMP) in urine and K. pneumoniae (IMP) in blood (90%). Clinical diagnostics from rectal swabs: 143 clinical rectal swab transport-media samples (63 KPC-positive, 80 negative by hospital testing) were tested directly (2 µL sample + 18 µL ANCA mix; 75°C). A cut-off based on negative sample statistics (T0–T=10.1) differentiated positives from negatives, with strong correlation between ANCA T0–T and PCR Ct. To satisfy regulatory sample size expectations, 57 additional mock positives were prepared by spiking KPC into negative transport media, yielding 200 total tests with KPC circuit sensitivity 100% and specificity 98.7%; ROC AUC was reported as 0.999. For IMP, 160 specimens (80 negative clinical, 80 spiked positives) produced 100% sensitivity and specificity with AUC=1.0. Direct rectal swab testing: Cotton swabs were used directly in the ANCA reaction (no transport media). The KPC circuit achieved sensitivity and specificity of 100% each. Comparing T0–T values indicated approximately 58-fold higher apparent target concentration on direct swabs vs transport media, consistent with dilution effects. Surface capture with 3D nanopillar swabs: A 3D nanopillar array swab was fabricated via replication and nanowire growth on metallized nanopillar arrays mounted on a 3D-printed backbone. The swab efficiently captured bacteria from pig skin and inanimate surfaces (desk, glove, scissors, knob, tweezers). SEM confirmed capture. Swab pieces were directly incubated with ANCA mixes; fluorescence increased only when the corresponding CPKP strain was present, demonstrating on-site capture and identification. Experiments were independently replicated three times. Statistics and ethics: Sample sizes followed KFDA guidance; investigators were blinded for clinical testing. The study had IRB approval (Gyeongsang National University Changwon Hospital, 2022-10-012).

- ANCA enables one-step, amplification-free, isothermal detection of target DNA at 75°C by integrating Ago-mediated cleavage within a positive feedback nucleic acid circuit. - Analytical performance: For KPC targets, threshold time correlated linearly with log concentration (R²=0.99) over 10 fM–10 nM with a reported LOD of 1.87 fM. For IMP, linearity 1 fM–1 nM with LOD 17.8 fM (R²=0.99). Additional targets (VIM, NDM, OXA-48) showed linearity from 1 fM–1 nM with reported LODs of 529 nM, 120 nM, and 144 nM, respectively. - Component controls confirmed low background without target, Ago, or guides; absence of R* eliminated amplification, validating the positive feedback design. - Direct detection of cultured antibiotic-resistant bacteria (K. pneumoniae KPC/IMP) without lysis or extraction showed specific identification by corresponding circuits; VIM, NDM, and OXA-48 circuits also discriminated resistant strains from wild type. - In spiked clinical matrices, KPC circuits detected K. pneumoniae in up to 99% of urine samples and 90% of blood samples; IMP circuits detected targets in urine and blood at high rates. - Clinical diagnostics from rectal swab transport media: Among 200 total tests (clinical + spiked), KPC detection achieved 100% sensitivity and 98.7% specificity with ROC AUC reported as 0.999; IMP detection (160 tests) achieved 100% sensitivity and 100% specificity with AUC=1.0. - Direct rectal swab testing achieved 100% sensitivity and 100% specificity and showed roughly 58-fold higher apparent target concentration compared with transport media, consistent with dilution effects. - 3D nanopillar swab integrated with ANCA enabled capture and on-site detection of CPKP from diverse contaminated surfaces; fluorescence increased only for matched target strains, with SEM verification of capture. - The assay relies on a single protein (Ago) and DNA guides, reducing complexity and cost, and does not require PAM motifs, enhancing target flexibility.

The study demonstrates that embedding Argonaute’s programmable cleavage into an artificial nucleic acid circuit produces autonomous, isothermal signal amplification sufficient for direct detection of carbapenemase-producers without nucleic acid extraction or PCR. The positive feedback loop (via T1/T2 triggers) amplifies signals from minimal target inputs, addressing sensitivity limitations of previous amplification-free Ago assays. By avoiding PAM dependence and using stable, cost-effective DNA guides, ANCA broadens target selection and improves practicality versus many CRISPR diagnostics. The method maintained performance in complex matrices (urine, blood) and on real-world clinical specimens (rectal swabs), correlating well with PCR results. Integration with 3D nanopillar swabs further extends the approach to on-site surface surveillance, capturing bacteria efficiently and enabling immediate detection. Collectively, these results support ANCA as a rapid, simple, and accurate tool for CPE surveillance and infection control, potentially reducing diagnostic turnaround and mitigating nosocomial spread.

This work introduces ANCA, a one-step, amplification-free, isothermal diagnostic that harnesses Argonaute-driven positive feedback to detect carbapenemase genes with high specificity and sensitivity directly from clinical and environmental samples. The platform detects multiple resistance targets (KPC, IMP, VIM, NDM, OXA-48), functions robustly in crude matrices, and achieves excellent diagnostic performance on rectal swabs, with further utility when paired with 3D nanopillar swabs for on-site capture. Future research should optimize Ago/guide efficiency to lower LODs across targets, expand validation with prospectively collected clinical positives (e.g., IMP), streamline hardware for point-of-care deployment, and broaden panels for multiplexed resistance surveillance.

- Reported LODs across targets are inconsistent and, for some targets (e.g., VIM, NDM, OXA-48), relatively high compared with leading amplified nucleic acid tests, reflecting early-stage optimization. - The assay operates at 75°C, which may pose constraints for some point-of-care settings and requires temperature-controlled devices. - Clinical IMP-positive rectal swabs were unavailable; IMP performance was assessed using spiked (mock) positives rather than confirmed clinical positives. - While specificity was high, one false positive was observed in transport-media testing for KPC. - The approach was validated on selected organisms and genes; broader inclusivity/exclusivity panels and evaluation against diverse clinical strains are needed to confirm generalizability.