Probe-free label system for rapid detection of Cronobacter genus in powdered infant formula

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.

Keywords: Cronobacter species; Loop-mediated isothermal amplification; Powdered infant formula; Probe-free label based lateral flow assay.

Fig. 1

Schematic diagram of the lateral flow assay. a Schematic view of lateral flow assay and the detection principle; b illustration of LFA test results

Fig. 2

The analysis of the primer dimers’ location in loop-mediated isothermal amplification assay. a Agarose gel electrophoresis analysis; b lateral flow assay analysis

Fig. 3

The optimization of Taq SSB protein. M marker. Lanes 1–8: the concentrations of Taq SSB protein ranging from 0 to 7 ng/μL. SSB single strand DNA binding

Fig. 4

The analysis of the influence of Taq SSB protein in the LAMP reaction. a Agarose gel electrophoresis analysis; b lateral flow assay analysis. SSB = Single strand DNA binding

Fig. 5

The sensitivity test of Cronobacter spp. in pure culture. a PCR; b loop-mediated isothermal amplification-agarose gel electrophoresis (LAMP-AGE); c loop-mediated isothermal amplification-lateral flow assay (LAMP-LFA). M: marker. Lanes 1 to 8: the concentrations of Cronobacter sakazakii ATCC 29544 ranging from 4.8 × 10⁶ to 4.8 × 10⁻¹ cfu/mL; lane 9, negative control

Fig. 6

The sensitivity test of Cronobacter spp. in PIF. a PCR; b loop-mediated isothermal amplification-agarose gel electrophoresis (LAMP-AGE); c loop-mediated isothermal amplification-lateral flow assay (LAMP-LFA). M: marker. Lanes 1 to 6: the PIF contaminated with concentrations of the Cronobacter sakazakii ATCC 29544 ranging from 5.6 × 10⁵ to 5.6 × 10⁰ cfu/g. Lane 7, negative control