Ultrasensitive Hybridization Chain Reaction-Assisted Multisite Exonuclease III Amplification Strategy Combined with a...

Area of Science:

• Molecular Biology
• Biotechnology
• Analytical Chemistry

Background:

• Accurate and timely bacterial detection is crucial for preventing environmental spread and disease outbreaks.
• Nucleic acid tests (NAT) are vital for early pathogen diagnosis, but often require complex laboratory setups.
• Isothermal amplification methods offer potential for simplified, on-site nucleic acid detection.

Purpose of the Study:

• To develop an ultrasensitive, one-pot isothermal amplification strategy for bacterial 16S rRNA.
• To integrate this strategy with a portable fluorescence detection device (PFD) and lateral flow assay (LFA) for direct signal readout.
• To enable simultaneous and specific detection of multiple bacterial species for on-site applications.

Main Methods:

• A hybridization chain reaction (HCR)-mediated, multisite exonuclease III (Exo-III) amplification strategy (HCR/Exo-III amplifier) was designed.
• Target-initiated HCR products triggered Exo-III amplification, generating numerous target amplicons.
• Amplicons were detected on a lateral flow assay (LFA) strip using DNA-CdTe/CdS probes and a portable fluorescence detection device (PFD).

Main Results:

• The HCR/Exo-III amplifier-based PFD-LFA achieved ultrasensitive detection of bacterial 16S rRNA.
• Detection limits were as low as tenths of fM for synthetic fragments and dozens of CFU/mL for live bacteria.
• Simultaneous and specific detection of *Staphylococcus aureus*, *Listeria monocytogenes*, and *Salmonella typhimurium* was demonstrated.

Conclusions:

• The developed sensing platform offers isothermal amplification, convenient operation, and good economy.
• This method shows significant potential for on-site testing of multiple nucleic acid analytes.
• The HCR/Exo-III amplifier-based PFD-LFA provides a promising tool for rapid bacterial detection in various settings.