Simple Screening of Listeria monocytogenes Based on a Fluorescence Assay via a Laminated Lab-On-Paper Chip

Abstract

Monitoring food safety is essential for protecting the health and safety of consumers. Conventional methods used are time consuming and laborious, requiring anywhere from three to seven days to obtain results. Thus, better monitoring methods are required. In this study, a laminated lab-on-paper chip was developed, and its use for the screening of ready-to-eat seafood was demonstrated. The assay on a chip was based on loop-mediated isothermal DNA amplification (LAMP) of the hly gene of Listeria monocytogenes and fluorescence signal detection via SYBR Gold^TM. Overall assay processes were completed in 4.5 h., (including 3.5 h. incubation for the bacteria enrichment, direct DNA amplification with no DNA extraction, and signal detection), without relying on standard laboratory facilities. Only positive samples induced fluorescence signals on chip upon illumination with UV light (λ = 460). The method has a limit of detection of 100 copies of L. monocytogenes DNA per 50 g of sample. No cross-reactivity was observed in samples contaminated with other bacteria. On-site monitoring of the seafood products using this chip revealed that one of 30 products from low sanitation vendors (3.33%) were contaminated, and these agreed with the results of PCR. The results demonstrated a benefit of this chip assay for practical on-site monitoring.

Keywords: Detection; LAMP; Lab-on-paper chip; Listeria monocytogenes; frozen seafood; hly gene.

Figure 1

Design of the chip and its structure. The chip was divided into two parts, one for DNA amplification (R) and the other for detection (D). Both were connected with a channel made of fishing line. The LAMP reagent and DNA sample were introduced via inlet 1, and the blue AgNPLs/probe was introduced via inlet 2. DNA amplification occurred after sealing the inlets with PE stickers and incubating at 63 °C for 30 min. For DNA detection, the amplified products were squeezed to touch a volume adjusting pad first (arrow 1); later, the buffer solution was squeezed through to collect the DNA from the volume adjusting pad (arrow 2), spun down, mixed, and placed on a UV light source. The fluorescence in a positive sample could be visualized by the naked eye.

Figure 2

DNA products from the amplification of the hly gene by LAMP compared with the PCR products and with detection on a chip; Lane M, 100 base pair ladder; Lane ntc, non-template control; and Lane P, positive DNA from Listeria monocytogenes.

Figure 3

The specificity of the LAMP reaction for Listeria monocytogenes compared with that of the PCR and detection on the chip; Lane 1, 100 base pair ladder; Lane 2, non-template control; Lane 3, L. monocytogenes plasmid; Lanes 4–10, seven strains of L. monocytogenes DNA; Lanes 11–14, four strains of L. innocua; Lanes 15–16, two strains of L. ivanovii; and Lanes 17–25, DNA from L. welshimeri, Vibrio cholera, V. parahaemolyticus, Salmonella enteritidis, Escherichia coli O157:H7, E. coli ETEC, E. coli EPEC, Pseudomonas putida, and Shigella flexneri.

Figure 4

(a) The detection limit of the LAMP reaction using DNA as the template compared with that of the PCR and the on-chip assay; Lane 1, 100 base pair ladder; Lane 2, non-template control; Lanes 3–11, 10-fold serial dilutions of L. monocytogenes template DNA, ranging from 10⁷ copies to 0 copies. (b) The detection limit by spiking the seafood sample with Listeria monocytogenes diluents with Lanes 15–19, from 10¹⁰ CFU to 10⁶ CFU; Lane 20, 1.02 × 10⁵ CFU; Lane 21, 10250 CFU; Lane 22, 1033 CFU; Lane 23, 100 CFU; Lane 24, 7 CFU; and Lane 25, 0 CFU. Lane 12, 100 base pair ladder; Lane 13, non-template control; and Lane 14, L. monocytogenes plasmid.