Integrating Recombinase Polymerase Amplification and Photosensitization Colorimetric Detection in One Tube for Fast Screening of C. sakazakii in Formula Milk Powder

Abstract

Cronobacter sakazakii (C. sakazakii) is a widely existing opportunistic pathogen and thus threatens people with low immunity, especially infants. To prevent the outbreak, a rapid and accurate on-site testing method is required. The current standard culture-based method is time-consuming (3−4 days), while the nucleic acid amplification (PCR)based detection is mostly carried out in central laboratories. Herein, isothermal recombinase polymerase amplification (RPA) coupled with a photosensitization colorimetric assay (PCA) was adopted for the on-site detection of C. sakazakii in powdered infant formulas (PIFs). The lowest visual detection concentration of C. sakazakii is 800 cfu/mL and 2 cfu/g after 8 h bacteria pre-enrichment. Furthermore, to avoid typical cap opening-resulted aerosol pollution, the PCA reagents were lyophilized onto the cap of the RPA tube (containing lyophilized RPA reagents). After amplification, the tube was subjected to simple shaking to mix the PCA reagents with the amplification products for lightdriven color development. Such a one-tube assay offered a lowest concentration of 1000 copies of genomic DNA of C. sakazakii within 1 h. After 8 h of bacterial enrichment, the lowest detecting concentration could be pushed down to 5 cfu/g bacteria in PIF. To facilitate on-site monitoring, a portable, battery-powered PCA device was designed to mount the typical RPA 8-tube strip, and a color analysis cellphone APP was further employed for facile readout.

Photoelectrochemical/Colorimetric Dual-Mode Specific Detection of Staphylococcus aureus Based on the Enzymatic Reaction Triggered by Catalase from Lysed Bacteria

“…Traditional methods, such as plate counting and polymerase Chain Reaction (PCR), often require considerable time, financial investment, and complex procedures. Currently, biosensors have emerged as a promising alternative to conventional methods for the detection of bacteria, including electrochemical detection, colorimetry, surface-enhanced Raman scattering (SERS), photoelectric chemical detection (PEC), fluorescence sensing, etc. Among them, PEC biosensors are considered a promising strategy due to the detection signal and excitation signal in different energy forms, offering lower background signals and enhanced sensitivity .…”