In this study, a whole-course nucleic-acid-constructed biosensor that combines the all-in-one concepts of the universal blocking linker recombinase polymerase amplification (UBLRPA) and a peptide nucleic acid (PNA)-based lateral flow device (PLFD) has been developed for the ultrasensitive detection of food pathogens. Using the preamplification UBLRPA principle, a universal linker and C3 space blocker were utilized to produce the universal linker single-duplex DNA products. The developed amplification system was employed to convert duplex products to a single strand. In the signal recognition strategy, a special PNA probe was successfully employed in the portable PLFD. The UBLRPA products were identified visually using the PLFD through dual hybridization (a PNA probe on the gold nanoparticle (Au-NP) was combined with a universal linker on the end of the products; a PNA capture probe was used on the test line and a universal linker on the other end of the products). The accumulation of Au-NPs produced a characteristic red band, enabling the visual detection of a food pathogen without further testing. To demonstrate the value of the all-in-one biosensor, Salmonella enterica subsp. enterica serovar typhimurium was used as a model organism. The biosensor showed high selectivity and extraordinary repeatability using S. typhimurium, and the limit of detection was 4 CFU mL. Furthermore, when milk samples artificially contaminated with S. typhimurium were analyzed, the analysis was completed within 30 min without complicated instrumentation. The results exhibited good precision and recovery. This portable all-in-one biosensor demonstrates great promise for the screening of pathogens in food and environmental samples.
Recent outbreaks of life-threatening neonatal infections linked to Enterobacter sakazakii (ES) heightened the need to develop rapid and ultrasensitive detection strategies, especially those capable of determining the viable cells. This study introduced a continual cascade nanozyme biosensor for the detection of viable ES based on propidium monoazide (PMA), loop-mediated isothermal amplification (LAMP), and Nanozyme strip. The ompA gene of ES was determined using FITC-modified and BIO-modified primers in the LAMP process. LAMP combined with PMA treatment was applied for distinguishing the viable from the dead state of ES. Then, using FeO magnetic nanoparticles as a nanozyme probe, a magnetic nanoparticle (MNP)-based immunochromatographic strip (Nanozyme strip) was further employed for amplifying signal to allow visual detection and quantification by a strip reader. The LAMP products were sandwiched between the anti-FITC and the anti-BIO, and the accumulation of the FeO magnetic nanoparticles enabled the visual detection of ES. The detection limit of the nanozyme biosensor was improved by 10 CFU/mL compared with previously reported techniques, and the whole manipulation process was much faster (within 1 h) and simpler (without specialist facilities). Hence, the developed continual cascade nanozyme biosensor has provided a rapid, ultrasensitive, and simple tool for on-site detection of viable ES.
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