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

Endonucleases have recently widely used in molecular diagnostics. Here, we report a strategy to exploit the properties of Argonaute (Ago) proteins for molecular diagnostics by introducing an artificial nucleic acid circuit with Ago protein (ANCA) method. The ANCA is designed to perform a continuous autocatalytic reaction through cross-catalytic cleavage of Ago protein, enabling one-step, amplification-free, and isothermal DNA detection. Using the ANCA method, carbapenemase-producing Klebsiella pneumoniae (CPKP) are successfully detected without additional reaction steps. In addition, we demonstrated the detection of carbapenem-resistant bacteria in human urine and blood samples using the method. We also demonstrate the direct identification of CPKP swabbed from surfaces using the ANCA method in conjunction with a three-dimensional nanopillar structure. Finally, the ANCA method is applied to detect CPKP in renal wash specimens from infected patients, achieving sensitivity and specificity of 100% and 100%, respectively. The developed method can contribute to simple, rapid and accurate diagnosis of CPKP, which can help prevent nosocomial infections.