Enzyme-Assisted Endogenous Guide DNA Generation-Mediated Pyrococcus furiosus Argonaute for Alicyclobacillus acidoterrestris Detection

Pyrococcus furiosus Argonaute ( Pf Ago) emerged as a novel endonuclease for the nucleic acid test recently. However, the input of exogenous guide DNA (gDNA) to activate Pf Ago has reduced its flexibility. In this work, an enzyme-assisted e ndogenous g DNA g eneration-mediated P f A go for the target d etection strategy, termed EGG-PAD, was proposed. With the aid of EcoR Ι, the target double-strand DNA was cut, producing a phosphate group at the 5′ end, functioning as gDNA to activate Pf Ago for nucleic acid detection. The applicability of this assay was tested in the detection of Alicyclobacillus acidoterrestris, a bacterium causing the spoilage of fruit juice, showing excellent sensitivity and specificity, ascribed to the “duplex amplification and triple insurance” mechanism. Moreover, EGG-PAD exhibited superior versatility in the identification of common foodborne pathogens. This powerful platform could also be an on-site test tool for detecting nucleic acid-containing organisms such as tumor cell, pathogen, and virus in the future.