Rapid and Sensitive Detection of Shigella spp. and Salmonella spp. by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique

Abstract

Shigella and Salmonella are frequently isolated from various food samples and can cause human gastroenteritis. Here, a novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully established and validated for simultaneous detection of Shigella strains and Salmonella strains in only a single reaction. Two sets of MERT-LAMP primers for 2 kinds of pathogens were designed from ipaH gene of Shigella spp. and invA gene of Salmonella spp., respectively. Under the constant condition at 63°C, the positive results were yielded in as short as 12 min with the genomic DNA extracted from the 19 Shigella strains and 14 Salmonella strains, and the target pathogens present in a sample could be simultaneously identified based on distinct fluorescence curves in real-time format. Accordingly, the multiplex detection assay significantly reduced effort, materials and reagents used, and amplification and differentiation were conducted at the same time, obviating the use of postdetection procedures. The analytical sensitivity of MERT-LAMP was found to be 62.5 and 125 fg DNA/reaction with genomic templates of Shigella strains and Salmonella strains, which was consist with normal LAMP assay, and at least 10- and 100-fold more sensitive than that of qPCR and conventional PCR approaches. The limit of detection of MERT-LAMP for Shigella strains and Salmonella strains detection in artificially contaminated milk samples was 5.8 and 6.4 CFU per vessel. In conclusion, the MERT-LAMP methodology described here demonstrated a potential and valuable means for simultaneous screening of Shigella and Salmonella in a wide variety of samples.

Keywords: LAMP; LoD; MERT-LAMP; Salmonella spp.; Shigella spp..

Figure 1

Sequence and location of Shigella (ipaH) and Salmonella (invA) genes used to design MERT-LAMP primers. The nucleotide sequences of the sense strands of ipaH (A) and invA (B) are listed. The sites of primer sequences were underlined. Left arrows and right arrows showed complementary and sense sequences that are used.

Figure 2

Confirmation and detection of Shigella- and Salmonella-LAMP products. (A,B) Color change of Shigella- and Salmonella-LAMP tubes; tube 1, positive amplification; tube 2, negative amplification. (C,D), 2% agarose gel electrophoresis applied to Shigella- and Salmonella-LAMP products; lane 1, DL 100-bp DNA marker; lane 2, positive LAMP reaction, lane 3, negative LAMP reaction.

Figure 3

Sensitivity of the ipaH- and invA-LAMP assays using serially genomic DNA with Shigella strains and Salmonella strains as templates. Sensitivity of ipaH-LAMP (A) and invA-LAMP (B) for Shigella and Salmonella detection was analyzed by real-time measurement of turbidity and the corresponding curves of concentrations of genomic DNA were marked in the figure. The LoD of ipaH-LAMP assay was 62.5 fg per tube, and the invA-LAMP for 125 fg per reaction. Sensitivity of ipaH-LAMP (C) and invA-LAMP (D) for Shigella and Salmonella detection was monitored by 2% agarose gel electrophoresis, and the positive amplifications were seen as a ladder-like pattern on 2% agarose gel electrophoresis analysis. Lane 1, DL 100-bp DNA marker.

Figure 4

Confirmation and detection of ipaH- and invA-MERT-LAMP products. (A,B) Color change of ipaH- and invA-MERT-LAMP tubes; tube 1, positive amplification; tube 2, negative amplification. (C,D) 2% agarose gel electrophoresis applied to ipaH- and invA-MERT-LAMP products; lane 1, DL 100-bp DNA marker; lane 2, positive LAMP reaction, lane 3, negative LAMP reaction.

Figure 5

Sensitivity of the ipaH- and invA-MERT-LAMP assays using serially genomic DNA with Shigella strains and Salmonella strains as templates. Sensitivity of ipaH-MERT-LAMP (A) and invA-MERT-LAMP (B) for Shigella and Salmonella detection was monitored by real-time format, and signals 1, 2, 3, 4, 5, 6, 7, 8, and 9 represent DNA levels of 2.5 ng, 250 pg, 25 pg, 2.5 pg, 250 fg, 125 fg, 62.5 fg, and 31.25 fg per vessel and negative control. The LoD of ipaH-MERT-LAMP assay was 62.5 fg per tube, and the invA-MERT-LAMP for 125 fg per reaction. Sensitivity of ipaH-MERT-LAMP (C) and invA-MERT-LAMP (D) for Shigella and Salmonella detection was analyzed by 2% agarose gel electrophoresis, and the positive amplifications were observed as a ladder-like pattern on 2% agarose gel electrophoresis analysis. Lane 1, DL 100-bp DNA marker.

Figure 6

The optimal temperature for multiplex MERT-LAMP assay. Two sets of MERT-LMAP primers targeting ipaH and invA genes were used in the same reaction tube, (A,B) were simultaneously obtained from Cy5 (labeling Shi-EFIP of ipaH) and Hex (labeling Sal-EFIP of invA) channels, respectively. The multiplex MERT-LAMP amplifications were detected by means of real-time format, and the corresponding curves of DNA concentrations were listed. Signal 1 indicates Shigella flexneri strains of in Cy5 channel (A), Salmonella Enteritidis strain for Hex channel (B), and signal 2 indicates negative control. Eight kinetic graphs (1–8) were generated at different amplification temperature (60°C–67°C) with Shigella genomic DNA at the level of 2.5 ng in Cy5 channel (A); another eight kinetic graphs (1–8) were yielded at different detection temperature (60°C–67°C) with Salmonella genomic DNA at the level of 2.5 ng in Hex channel (B). The graphs from 62°C to 65°C show robust amplification.

Figure 7

The sensitivity of multiplex MERT-LAMP assay for simultaneously detecting two target pathogens. Two sets of MERT-LMAP primers targeting ipaH and invA genes were simultaneously added to a reaction tube. (A,B) were simultaneously generated from Cy5 (labeling Shi-EFIP of ipaH) and Hex (labeling Sal-EFIP of invA) channels, respectively. Sensitivity of multiplex-MERT-LAMP for simultaneously detecting Shigella (A) and Salmonella (B) was monitored by real-time format, and signals 1, 2, 3, 4, 5, 6, 7, 8, and 9 represent DNA levels of 2.5 ng, 250 pg, 25 pg, 2.5 pg, 250 fg, 125 fg, 62.5 fg, and 31.25 fg per vessel and negative control. The LoD of multiplex MERT-LAMP assay for Shiglla detection was 62.5 fg per tube, and the LoD of multiplex MERT-LAMP for Salmonella detection was 125 fg per reaction.

Figure 8

The specificity of multiplex MERT-LMAP detection for different strains. The multiplex MERT-LAMP reactions were conducted using different genomic DNA templates and were analyzed by means of real-time format. (A,B) were simultaneously yielded from Cy5 and Hex channels. Signals 1–8, Shigella flexneri strains of serovar 1d (ICDC-NPS001), 4a (ICDC-NPS002), 5a (ICDC-NPS003), 2b (ICDC-NPS004), 1b (ICDC-NPS005), Shigella boydii, Shigella sonneri and Shigella dysenteriae; signals 9–13, Salmonella Choleraesuis (ICDC-NPSa001), Salmonella Dublin (ICDC-NPSa002), Salmonella Enteritidis (ICDC-NPSa003), Salmonella Typhimurium (ICDC-NPSa004), Salmonella Weltevreden (ICDC-NPSa005); signals 14 and 15, two Salmonella strains of unidentified serotype; signals 16–35, Listeria monocytogenes stains of serovar 1/2a (EGD-e), Listeria monocytogenes stains of serovar 4a (ATCC19114), Enteropathogenic E. coli, Enterotoxigenic E. coli, Enteroaggregative E. coli, Enteroaggregative E. coli, Enteroinvasive E. coli, Enterohemorrhagic E. coli, Plesiomonas shigelloides, Enterobacter cloacae, Enterococcus faecalis, Yersinia enterocolitica, Bntorobater sakazakii, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Campylobacter jejuni, Pseudomonas aeruginosa, and Bacillus cereus, signal 36, negative control.

Figure 9

The sensitivity of multiplex MERT-LAMP assay for simultaneously detecting two target pathogens in artificially contaminated milk samples. Two sets of MERT-LMAP primers targeting ipaH and invA genes were added to a reaction tube. (A,B) were simultaneously generated from Cy5 (labeling Shi-EFIP of ipaH) and Hex (labeling Sal-EFIP of invA) channels, respectively. Sensitivity of multiplex-MERT-LAMP for simultaneously detecting Shigella (A) and Salmonella (B) in artificially contaminates milk samples was analyzed by real-time format, and signals 1, 2, 3, 4, 5, 6, and 7 represent Shigella DNA levels of 5800, 580, 58, 5.8, 0.58, and 0.058 CFU per reaction and negative control; Salmonella DNA levels for 6400, 640, 64, 6.4, 0.64, and 0.064 CFU per reaction and negative control. The LoD of multiplex MERT-LAMP assay for Shiglla detection in artificially contaminates milk samples was 5.8 CFU per tube, and the LoD of multiplex MERT-LAMP for Salmonella detection in artificially contaminates milk samples was 6.4 CFU per reaction.