The Novel Multiple Inner Primers-Loop-Mediated Isothermal Amplification (MIP-LAMP) for Rapid Detection and Differentiation of Listeria monocytogenes

Abstract

Here, a novel model of loop-mediated isothermal amplification (LAMP), termed multiple inner primers-LAMP (MIP-LAMP), was devised and successfully applied to detect Listeria monocytogenes. A set of 10 specific MIP-LAMP primers, which recognized 14 different regions of target gene, was designed to target a sequence in the hlyA gene. The MIP-LAMP assay efficiently amplified the target element within 35 min at 63 °C and was evaluated for sensitivity and specificity. The templates were specially amplified in the presence of the genomic DNA from L. monocytogenes. The limit of detection (LoD) of MIP-LAMP assay was 62.5 fg/reaction using purified L. monocytogenes DNA. The LoD for DNA isolated from serial dilutions of L. monocytogenes cells in buffer and in milk corresponded to 2.4 CFU and 24 CFU, respectively. The amplified products were analyzed by real-time monitoring of changes in turbidity, and visualized by adding Loop Fluorescent Detection Reagent (FD), or as a ladder-like banding pattern on gel electrophoresis. A total of 48 pork samples were investigated for L. monocytogenes by the novel MIP-LAMP method, and the diagnostic accuracy was shown to be 100% when compared to the culture-biotechnical method. In conclusion, the MIP-LAMP methodology was demonstrated to be a reliable, sensitive and specific tool for rapid detection of L. monocytogenes strains.

Keywords: HlyA gene amplification; L. monocytogenes detection; LAMP; LoD; MIP-LAMP.

Figure 1

The principle of MIP-LAMP amplification. The schematic showing the mechanism of the novel MIP-LAMP assay.

Figure 2

Primers design of MIP-LAMP. (A), location and orientation of L. monocytogenes specific MIP-LAMP primers within the nucleotide sequence of the listeriolysin O (hlyA) gene (GenBank accession number M24199). The nucleotide sequences of the primer sites are underlined, left and right arrows indicate complementary and sense sequences that are used; (B), diagram exhibiting the positions at which the primers attach to amplify the target sequence.

Figure 3

Amplification products of 4 nLAMP assays were visually detected by FD. (A–D): nLAMP assay using primers set G1, G2, G3 and G4, respectively. Tube 1, positive amplification, tube 2, negative amplification.

Figure 4

Agarose gel electrophoresis of 4 nLAMP products. (A–D): nLAMP assay using primers set G1, G2, G3 and G4, respectively. Lane 1, DL 50-bp DNA marker; lane 2, positive nLAMP products; lane 3 negative control.

Figure 5

Amplification products of MIP-LAMP assays were visually detected both by FD and agarose gel electrophoresis. (A): color change of MIP-LAMP tubes; tube 1, positive amplification; tube 2, negative amplification; (B): agarose gel electrophoresis of MIP-LAMP products; lane 1, DL 50-bp DNA marker; lane 2, positive nLAMP products; lane 3 negative control.

Figure 6

Sequencing analysis of MIP-LAMP amplified L. monocytogenes hlyA gene. The target sequences were presented at the top and the sequences of the primers sites (P1, P2, B1 and B2) were underlined. (A–D): the sequencing data were obtained from primers P1 and B1, P1 and B2, P2 and B1, P2 and B2, respectively. Left and right arrows indicated complementary and sense sequences that were used. The sequencing data were shown in the bottom.

Figure 7

The optimal temperature for the MIP-LAMP assay. The MIP-LAMP amplifications reactions were analyzed by real-time measurement of turbidity and the corresponding curves of concentrations of DNA were marked in the Figure. The threshold value was 0.1 and the turbidity of >0.1 was considered to be positive. Eight kinetic graphs (A–H) were obtained at different temperature (60–67 °C) with L. monocytogenes DNA at the level of 2.5 pg per reaction, and the graphs from B to F showed robust amplification.

Figure 8

Products of MIP-LAMP monitored using 2.5% agarose gel electrophoresis. The products (A–H) of MIP-LAMP from different reaction temperature (60–67 °C) were monitored by 2.5% agarose gel electrophoresis after staining with ethidium bromide. Lane M, DL 100-bp DNA marker; lane 1, positive MCDA products; lane 2, negative control (no DNA).

Figure 9

Specificity of MIP-LAMP detection for different strains. Lane M, DL 50-bp DNA marker; lane 1–12, L. monocytogenes strains of serovar 1/2a (EGD-e), 3a (ICDCLM023), 1/2c (ICDCLM010), 3c (ICDCLM446), 1/2b (ICDCLM007), 3b (ICDCLM078), 7 (NCTC10890), 4a (ATCC19114), 4c (ATCC19116), 4b (ICDC419), 4d (ATCC19117) and 4e (ATCC19118); lane 13–17, other Listeria reference strains of L. ivanovii (ATCCBAA-678), L. innocua (ATCCBAA-680), L. grayi (ATCC25402), L. seeligeri (ATCC35967), L. welshimeri (ATCC35897); lane 18–34, non-Listeria strains of Bacillus cereus, Enteropathogenic E. coli, Enterotoxigenic E. coli, Enteroaggregative E. coli, Enteroinvasive E. coli, Enterohemorrhagic E. coli, Plesiomonas shigelloides, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterococcus faecalis, Yersinia enterocolitica, Aeromonas hydrophila, Citrobacter freumdii, Proteus vulgaris, Vibrio fluvialis, Salmonella enterica.

Figure 10

Sensitivity of the MIP-LAMP and nLAMP assays using serially diluted genomic DNA with L. monocytogenes strain EGD-e as template. A, Sensitivity of MIP-LAMP (A1) and nLAMP (A2) for L. monocytogenes detection was analyzed by real-time measurement of turbidity. The LoD for MIP-LAMP and nLAMP assays were 62.5 fg and 250 fg genomic DNA per reaction, respectively; (B), Sensitivity of MIP-LAMP (B1) and nLAMP (B2) approaches for L. monocytogenes detection were seen by gel electrophoresis, respectively. Lane M, DL 50-bp DNA marker. The positive results were observed as a ladder-like pattern on 2.5% agarose gel electrophoresis analysis.