Rapid, sensitive, and accurate identification of pathogens is vital for preventing and controlling fish disease, reducing economic losses in aquaculture, and interrupting the spread of food-borne diseases in human populations. Herein, we proposed a hybridization chain reaction (HCR) cascaded dual-signal amplification platform for the ultrasensitive and specific detection of pathogenic microorganisms. A couple of specific primers for target bacterial 16S rRNAs were used to obtain amplified target single-stranded DNAs (AT-ssDNA). Then, AT-ssDNA initiated HCR amplification along with the opening of fluorophore (FAM) and a quencher (BHQ1) labeled hairpin reporter probe (H1), and the FAM fluorescence signal recovered. The proposed strategy could achieve a detection limit down to 0.31 CFU/mL for Staphylococcus aureus (S. aureus), 0.49 CFU/mL for Escherichia coli (E. coli) in buffer, and a linear range from 1 to 1 × 106 CFU/mL for S. aureus, 1 to 1 × 107 CFU/mL for E. coli. Furthermore, this platform enabled sensitive and precise detection of pathogenic microorganisms in complex samples such as fish blood and different organ tissues (large intestine, gallbladder, heart, liver, ren, gill, skin), which shows great potential in disease prevention and control in aquatic products.
Keywords: Bacterial pathogen detection; HCR; Nucleic acid amplification.
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