Ultrasensitive Hybridization Chain Reaction-Assisted Multisite Exonuclease III Amplification Strategy Combined with a Direct Quantitative Fluorescence Lateral Flow Technique for Multiple Bacterial 16S rRNA Detection

Accurate and in-time detection of bacteria conduces to preventing their rapid spread around the environment, while a nucleic acid test (NAT) is a powerful tool for early diagnosis of pathogens. Herein, we propose a hybridization chain reaction (HCR)-mediated multisite exonuclease III (Exo-III) amplification strategy (HCR/Exo-III amplifier) to achieve the one-pot and ultrasensitive isothermal amplification of bacterial 16S rRNA and a portable fluorescence detection device (PFD) to directly read signals in a lateral flow assay (LFA). In detail, the target-initiated HCR products present multiple binding sites for triggering the Exo-III amplifier that produces numerous target amplicons. Following that, the target amplicons travel up on the strip and bridge between the DNA-CdTe/CdS probes and the capture DNA to form a positive fluorescence line. After that, the strip is inserted into the PFD to accomplish the fluorescence signal reading. The constructed HCR/Exo-III amplifier-based PFD-LFA implemented the simultaneous and specific detection of three bacteria with a detection limit of a few tenths of fM for synthetic 16S rRNA fragments and dozens of CFU/mL for Staphylococcus aureus, Listeria monocytogenes, and Salmonella typhimurium in pure cultures. The sensing platform features isothermal amplification, convenient operation, and good economy, displaying great potential for on-site testing toward multiple nucleic acid analytes.