A novel photoreactive DNA-binding dye for detecting viable Klebsiella pneumoniae in powdered infant formula

In addition to Cronobacter spp., Klebsiella pneumoniae is another opportunistic bacterial pathogen present in powdered infant formula (PIF) that can cause pneumonia, septicemia, and other diseases. In this study, a rapid and specific method based on a fluorescence probe was developed for detecting viable K. pneumoniae in PIF samples via the combination of recombinase-aided amplification (RAA) with thiazole orange monoazide (TOMA) dye (the TOMA–RAA assay hereafter). As a novel photosensitive DNA-intercalating dye, TOMA was used to penetrate bacterial cells, including both dead and viable cells, as verified by confocal laser scanning microscopy and fluorescent emission spectrometry. Importantly, the RAA assay exhibited good performance in detecting K. pneumoniae within 40 min at 39°C. Under optimal conditions, the TOMA–RAA assay can detect as low as 2.6 × 10³ cfu/mL of K. pneumoniae in pure culture and 2.3 × 10⁴ cfu/g of K. pneumoniae in spiked PIF sample. After 3 h of pre-enrichment, 3 × 10⁰ cfu/g of K. pneumoniae can be detected. Furthermore, the TOMA–RAA assay displayed an excellent anti-interference ability to nontarget bacteria. In short, the proposed method has great potential application for the rapid and accurate detection of viable K. pneumoniae in PIF.