Argonaute-DNAzyme tandem biosensing for highly sensitive and simultaneous dual-gene detection of methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA), a common zoonotic multidrug-resistant bacterium, puts a great threat to public health and food safety. Rapid and reliable detection of MRSA is crucial to guide effective patient treatment at early stages of infection and control the spread of MRSA infections. Herein, we developed a Simultaneous dual-gene and ulTra-sensitive detection for methicillin-resistant Staphylococcus aureus using Argonaute-DNAzyme tandem Detection (STAND). Simply, loop-mediated isothermal amplification (LAMA) was used for the amplification of the species-specific mecA and nuc gene, followed by STAND enabled by the sitespecific cleavage of programable Argonaute. The Argonaute-DNAzyme tandem reaction rendered a conceptually novel signal amplification and transduction module that was more sensitive (1 or 2 order of magnitude higher) than the original Argonaute-based biosensing. With the strategy, the target nucleic acid signals gene were dexterously converted into fluorescent signals. STAND could detect the nuc gene and mecA gene simultaneously in a single reaction with 1 CFU/mL MRSA and a dynamic range from 1 to 108 CFU/mL. This method was confirmed by clinical samples and challenged by identifying contaminated foods and MRSA-infected animals. This work enriches the arsenal of Argonaute-mediated biosensing and presents a novel biosensing strategy to detect pathogenic bacteria with ultra-sensitivity, specificity and on-site capability.