Abstract
Cronobacter species previously known as Enterobacter sakazakii poses high risks to neonates and infants. In this work a rapid detection method was developed which combined loop-mediated isothermal amplification with lateral flow assay for detection of Cronobacter species in powdered infant formula. The fast amplification reaction without betaine was established and capable of performing DNA replication within 25 min. Based on the novel probe-free labeling methods, we established a lateral flow assay to capture the specific loop-mediated isothermal amplification amplicons which were labeled with fluorescein isothiocyanate and biotin. And the final detection time of this system was within 40 min. The false positive results of the lateral flow assay induced by primer dimer tagged with fluorescein isothiocyanate and biotin were eliminated by Taq single strand DNA binding protein (4 ng/μL). Simultaneously, the efficiency of the fast loop-mediated isothermal amplification assay was achieved. By injection of Taq SSB into the amplification assay as a replacement for betaine, the novel probe-free method could detect Cronobacter species with high specificity and sensitivity at the detection limit in PIF of 101 cfu/g. Our overall strategy has excellent potential in the rapid diagnosis of Cronobacter species label-free by integrating loop-mediated isothermal amplification and lateral flow assay.
Highlights
Cronobacter species (Cronobacter spp.) isolated from plant-based food products, previously known as Enterobacter sakazakii, are foodborne pathogens that pose a high risk of infection to neonates as well as immunocompromised individuals causing meningitis, necrotizing enterocolitis, and bacteremia (Susan and Forsythe 2011), and the fatality rate is about 40–80% (Yan and Fanning 2015)
Further we found that by agarose gel electrophoresis (AGE) analysis all the 15 combination modes as the negative control without DNA templates gave the negative results and the probe-hybridization assay as the positive control with DNA template provided the positive result
Compared with AGE analysis, one combination mode of F3 and LF in loop-mediated isothermal amplification (LAMP) assay as the negative control gave an unexpected positive result by lateral flow assay (LFA) analysis (Fig. 2)
Summary
Cronobacter species (Cronobacter spp.) isolated from plant-based food products, previously known as Enterobacter sakazakii, are foodborne pathogens that pose a high risk of infection to neonates as well as immunocompromised individuals causing meningitis, necrotizing enterocolitis, and bacteremia (Susan and Forsythe 2011), and the fatality rate is about 40–80% (Yan and Fanning 2015). All Cronobacter spp., except C. condimenti, have been associated with human infections (Cruz-Córdova et al 2012). The traditional cultural and biochemical-based methods are tedious and require skilled personnel and time (Blažková et al 2009). Rapid methods are viewed as alternative means to detect and monitor Cronobacter spp. Nucleic acid amplification methods including PCR (Moraes and Maruniak 1997; Chen et al 2015), multiplex PCR (Gordon et al 2015; Zhang et al 2015), real-time PCR (Cecilia et al 2015; Mai et al 2015) have attracted the attention of a plethora of engineers and scientists due
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