Rapid and Sensitive Detection of Vibrio parahaemolyticus and Vibrio vulnificus by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique

Vibrio parahaemolyticus and Vibrio vulnificus are two marine seafood-borne pathogens causing severe illnesses in humans and aquatic animals. In this study, a recently developed novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully developed and evaluated for simultaneous detection of V. parahaemolyticus and V. vulnificus strains in only a single reaction. Two MERT-LAMP primer sets were designed to specifically target toxR gene of V. parahaemolyticus and rpoS gene of V. vulnificus. The MERT-LAMP reactions were conducted at 62 °C, and the positive results were produced in as short as 19 min with the genomic DNA templates extracted from the V. parahaemolyticus and V. vulnificus strains. The two target pathogens present in the same sample could be simultaneously detected and correctly differentiated based on distinct fluorescence curves in a real-time format. The sensitivity of MERT-LAMP assay was 250 fg and 125 fg DNA per reaction with genomic templates of V. parahaemolyticus and V. vulnificus strains, which was in conformity with conventional LAMP detection. Compared with PCR-based techniques, the MERT-LAMP technology was 100- and 10-fold more sensitive than that of PCR and qPCR methods. Moreover, the limit of detection of MERT-LAMP approach for V. parahaemolyticus isolates and V. vulnificus isolates detection in artificially-contaminated oyster samples was 92 CFU and 83 CFU per reaction. In conclusion, the MERT-LAMP assay presented here was a rapid, specific, and sensitive tool for the detection of V. parahaemolyticus and V. vulnificus, and could be adopted for simultaneous screening of V. parahaemolyticus and V. vulnificus in a wide variety of samples.


Introduction
Vibrio parahaemolyticus and Vibrio vulnificus, which are the halophilic bacteria commonly found in the estuarine, coastal, and marine environments, belong to the family Vibrionaceae and cause seafood-borne gastrointestinal disorders in humans [1]. Ingestion of raw or undercooked seafood with

Detection of toxR-and rpoS-LAMP Products
To confirm the availability of toxRand rpoS-LAMP primers, the LAMP reaction either for V. parahaemolyticus strains or V. vulnificus strains was processed in the absence or presence of genomic templates at 62˝C. The amplification products were analyzed by visual inspection using FD reagent, and the positive amplifications in V. parahaemolyticusand V. vulnificus-LAMP tubes directly saw color change from light gray to green within 1h incubation periods (Figure 1). The conventional LAMP products were also analyzed by 2% agarose gel electrophoresis, and positive results showed the typical ladder-like patterns on gel electrophoresis ( Figure 1). Thus, the toxRand rpoS-LAMP primers for V. parahaemolyticus and V. vulnificus detection were found good candidates for developing the MERT-LAMP assays.

Detection of toxR-and rpoS-LAMP Products
To confirm the availability of toxR-and rpoS-LAMP primers, the LAMP reaction either for V. parahaemolyticus strains or V. vulnificus strains was processed in the absence or presence of genomic templates at 62 °C. The amplification products were analyzed by visual inspection using FD reagent, and the positive amplifications in V. parahaemolyticus-and V. vulnificus-LAMP tubes directly saw color change from light gray to green within 1h incubation periods (Figure 1). The conventional LAMP products were also analyzed by 2% agarose gel electrophoresis, and positive results showed the typical ladder-like patterns on gel electrophoresis ( Figure 1). Thus, the toxR-and rpoS-LAMP primers for V. parahaemolyticus and V. vulnificus detection were found good candidates for developing the MERT-LAMP assays.

Sensitivity of V. parahaemolyticus-and V. vulnificus-LAMP Assay in Pure Culture
Sensitivity of conventional LAMP assays on V. parahaemolyticus and V. vulnificus were evaluated by analyzing products produced from the serial dilutions (2.5 ng, 250 pg, 25 pg, 2.5 pg, 250 fg, 125 fg, 62.5 fg, and 31.25 fg per microliter) of the V. parahaemolyticus and V. vulnificus genomic DNA templates in triplicate. As shown in Figure 2, the LAMP reactions were analyzed by real-time turbidity detection; the limit of detection (LoD) of V. parahaemolyticus-LAMP assay was 250 fg per tube (Figure 2A), and the V. vulnificus-LAMP assay for 125 fg per tube ( Figure 2B). Moreover, the final LAMP products were also detected by 2% agarose gel electrophoresis, and positive amplifications were observed as a ladder-like pattern ( Figure 2C,D). The LoD of the agarose gel electrophoresis detection for V. parahaemolyticus-and V. vulnificus-LAMP reactions was in complete conformity with turbidity measurements.

Detection of MERT-LAMP Products in Non-Real-Time Format
In order to demonstrate the utility of two MERT-LAMP primer sets, the MERT-LAMP assay either for V. parahaemolyticus strains or V. vulnificus strains was processed in the presence or absence of genomic DNA templates according to the standard MERT-LAMP condition. The MERT-LAMP products were detected by visual inspection using FD reagent, and the color change of positive MERT-LAMP reactions in V. parahaemolyticus-and V. vulnificus tubes from light gray to green were

Sensitivity of V. parahaemolyticus-and V. vulnificus-LAMP Assay in Pure Culture
Sensitivity of conventional LAMP assays on V. parahaemolyticus and V. vulnificus were evaluated by analyzing products produced from the serial dilutions (2.5 ng, 250 pg, 25 pg, 2.5 pg, 250 fg, 125 fg, 62.5 fg, and 31.25 fg per microliter) of the V. parahaemolyticus and V. vulnificus genomic DNA templates in triplicate. As shown in Figure 2, the LAMP reactions were analyzed by real-time turbidity detection; the limit of detection (LoD) of V. parahaemolyticus-LAMP assay was 250 fg per tube (Figure 2A), and the V. vulnificus-LAMP assay for 125 fg per tube ( Figure 2B). Moreover, the final LAMP products were also detected by 2% agarose gel electrophoresis, and positive amplifications were observed as a ladder-like pattern ( Figure 2C,D). The LoD of the agarose gel electrophoresis detection for V. parahaemolyticusand V. vulnificus-LAMP reactions was in complete conformity with turbidity measurements.

Detection of MERT-LAMP Products in Non-Real-Time Format
In order to demonstrate the utility of two MERT-LAMP primer sets, the MERT-LAMP assay either for V. parahaemolyticus strains or V. vulnificus strains was processed in the presence or absence of genomic DNA templates according to the standard MERT-LAMP condition. The MERT-LAMP products were detected by visual inspection using FD reagent, and the color change of positive MERT-LAMP reactions in V. parahaemolyticusand V. vulnificus tubes from light gray to green were directly observed by naked eyes within 1 h incubation periods ( Figure 3A). Furthermore, the MERT-LAMP products were also detected by 2% agarose gel electrophoresis, and the typical ladder-like patterns were observed but not in negative control ( Figure 3 The sensitivity of toxR-LAMP assay was 250 fg per reaction, and the rpoS-LAMP for 125 fg per reaction. Sensitivity of toxR-LAMP (C) and rpoS-LAMP (D) for V. parahaemolyticus and V. vulnificus 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.

Detection of MERT-LAMP Products in Non-Real-Time Format
In order to demonstrate the utility of two MERT-LAMP primer sets, the MERT-LAMP assay either for V. parahaemolyticus strains or V. vulnificus strains was processed in the presence or absence of genomic DNA templates according to the standard MERT-LAMP condition. The MERT-LAMP products were detected by visual inspection using FD reagent, and the color change of positive MERT-LAMP reactions in V. parahaemolyticus-and V. vulnificus tubes from light gray to green were directly observed by naked eyes within 1 h incubation periods ( Figure 3A). Furthermore, the MERT-LAMP products were also detected by 2% agarose gel electrophoresis, and the typical ladder-like patterns were observed but not in negative control ( Figure 3).

Figure 2.
Sensitivity of the toxRand rpoS-LAMP assays using serially-genomic DNA with Vibrio parahaemolyticus isolates and Vibrio vulnificus isolates as templates. LoD of toxR-LAMP (A) and rpoS-LAMP (B) for V. parahaemolyticus and V. vulnificus detection was monitored by real-time measurement of turbidity and the corresponding curves of concentrations of genomic DNA were marked in the figure. The sensitivity of toxR-LAMP assay was 250 fg per reaction, and the rpoS-LAMP for 125 fg per reaction. Sensitivity of toxR-LAMP (C) and rpoS-LAMP (D) for V. parahaemolyticus and V. vulnificus 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. directly observed by naked eyes within 1 h incubation periods ( Figure 3A). Furthermore, the MERT-LAMP products were also detected by 2% agarose gel electrophoresis, and the typical ladder-like patterns were observed but not in negative control ( Figure 3).

The Optimal Amplification Temperature of MERT-LAMP Approach
In order to test the optimal detection temperature, the MERT-LMAP amplifications were performed at distinct temperatures (60 to 67˝C) with V. vulnificus-MERT-LAMP primer set according to the standard MERT-LAMP reaction. The reference strain ATCC 27562 was selected as a positive control to examine the optimal temperature at the level of 250 pg of genomic DNA template per vessel. The results were detected by means of real-time format, and the typical kinetics graphs were yielded ( Figure 4). Each amplification temperature produced a robust signal corresponding to Cy5 channel, with the faster amplifications were obtained from reaction temperatures of 60 to 64˝C, which were considered as the reference temperature for MERT-LAMP test. The detection temperature of 62˝C was applied for the rest of MERT-LAMP reactions conducted in this study.

The Optimal Amplification Temperature of MERT-LAMP Approach
In order to test the optimal detection temperature, the MERT-LMAP amplifications were performed at distinct temperatures (60 to 67 °C) with V. vulnificus-MERT-LAMP primer set according to the standard MERT-LAMP reaction. The reference strain ATCC 27562 was selected as a positive control to examine the optimal temperature at the level of 250 pg of genomic DNA template per vessel. The results were detected by means of real-time format, and the typical kinetics graphs were yielded ( Figure 4). Each amplification temperature produced a robust signal corresponding to Cy5 channel, with the faster amplifications were obtained from reaction temperatures of 60 to 64 °C, which were considered as the reference temperature for MERT-LAMP test. The detection temperature of 62 °C was applied for the rest of MERT-LAMP reactions conducted in this study.

Sensitivity of MERT-LAMP Assay for a Single Target
In the present study, we tested the MERT-LAMP approach in a single target format by using separate amplification of toxR (V. parahaemolyticus-specific gene) and rpoS (V. vulnificus-specific gene) from an V. parahaemolyticus and V. vulnificus DNA templates in each reaction, respectively. The Hex and Cy5 fluorophores, which were used for yellow and red channels, were assigned to toxRand rpoS-MERT-LAMP primer sets, respectively. As shown in Figure 5, the release of quenching was seen as a robust increase of Cy5 and Hex signals, and positive amplifications were generated in approximately 19 min. The LoD of MERT-LAMP technique for independently analyzing toxR and rpoS genes was 250 fg and 125 fg of genomic DNA templates per tube, respectively ( Figure 5). Moreover, the final MERT-LAMP products were also analyzed by 2% agarose gel electrophoresis; the typical ladder-like patterns were visible in positive amplifications but not in negative reactions and control. The LoD of the real-time detection for V. parahaemolyticusand V. vulnificus-MERT-LAMP amplifications was completed accordance with agarose gel electrophoresis measurement.

Sensitivity of MERT-LAMP Assay for a Single Target
In the present study, we tested the MERT-LAMP approach in a single target format by using separate amplification of toxR (V. parahaemolyticus-specific gene) and rpoS (V. vulnificus-specific gene) from an V. parahaemolyticus and V. vulnificus DNA templates in each reaction, respectively. The Hex and Cy5 fluorophores, which were used for yellow and red channels, were assigned to toxR-and rpoS-MERT-LAMP primer sets, respectively. As shown in Figure 5, the release of quenching was seen as a robust increase of Cy5 and Hex signals, and positive amplifications were generated in approximately 19 min. The LoD of MERT-LAMP technique for independently analyzing toxR and rpoS genes was 250 fg and 125 fg of genomic DNA templates per tube, respectively ( Figure 5). Moreover, the final MERT-LAMP products were also analyzed by 2% agarose gel electrophoresis; the typical ladder-like patterns were visible in positive amplifications but not in negative reactions and control. The LoD of the real-time detection for V. parahaemolyticus-and V. vulnificus-MERT-LAMP amplifications was completed accordance with agarose gel electrophoresis measurement. The LoD of PCR, real-time PCR, conventional LAMP, and MERT-LAMP approaches on V. parahaemolyticus was 25 pg, 2.5 pg, 250 fg and 250 fg per reaction, and on V. vulnificus was 25 pg, 2.5 pg, 125 fg and 125 fg per reaction, respectively (Table 1, Figures 2 and 5). These results suggested that the sensitivity of MERT-LAMP assay for detecting a single target was 10-fold and 100-fold more sensitive than that of qPCR and PCR assays, while was identical with conventional LAMP technique. The LoD of PCR, real-time PCR, conventional LAMP, and MERT-LAMP approaches on V. parahaemolyticus was 25 pg, 2.5 pg, 250 fg and 250 fg per reaction, and on V. vulnificus was 25 pg, 2.5 pg, 125 fg and 125 fg per reaction, respectively (Table 1, Figures 2 and 5). These results suggested that the sensitivity of MERT-LAMP assay for detecting a single target was 10-fold and 100-fold more sensitive than that of qPCR and PCR assays, while was identical with conventional LAMP technique.

Sensitivity of MERT-LAMP Assay for Multiple Targets in a Reaction
To examine the capability of MERT-LAMP technique for simultaneously detecting V. parahaemolyticus and V. vulnificus in a single reaction, we slightly adjusted the amount of the primers on the base of standard MERT-LAMP reaction, and the multiplex MERT-LAMP amplifications were carried out at 62˝C for 60 min. Two different fluorescence curves were simultaneously obtained from multiplex MERT-LAMP reactions, which contained two complete primer sets and their corresponding genomic templates ( Figure 6). The MERT-LAMP methodology successfully detected V. parahaemolyticus and V. vulnificus in a single reaction, and provided a robust signal for each target. The LoD of MERT-LAMP assay for simultaneously detecting toxR and rpoS genes was 250 fg and 125 fg of each genomic DAN template per tube, respectively ( Figure 6). No difference of sensitivity was obtained between detecting multiple targets and a single target in a MERT-LAMP reaction.

Sensitivity of MERT-LAMP Assay for Multiple Targets in a Reaction
To examine the capability of MERT-LAMP technique for simultaneously detecting V. parahaemolyticus and V. vulnificus in a single reaction, we slightly adjusted the amount of the primers on the base of standard MERT-LAMP reaction, and the multiplex MERT-LAMP amplifications were carried out at 62 °C for 60 min. Two different fluorescence curves were simultaneously obtained from multiplex MERT-LAMP reactions, which contained two complete primer sets and their corresponding genomic templates ( Figure 6). The MERT-LAMP methodology successfully detected V. parahaemolyticus and V. vulnificus in a single reaction, and provided a robust signal for each target. The LoD of MERT-LAMP assay for simultaneously detecting toxR and rpoS genes was 250 fg and 125 fg of each genomic DAN template per tube, respectively ( Figure 6). No difference of sensitivity was obtained between detecting multiple targets and a single target in a MERT-LAMP reaction.

Analytical Specificity of the Multiplex MERT-LAMP Approach
In order to test the MERT-LAMP technique's specificity, the multiplex MERT-LAMP amplifications were conducted under the multiplex MERT-LAMP conditions described above with the purely genomic DNA templates extracted from 19 V. parahaemolyticus, 15 V. vulnificus, and 20 non-V. parahaemolyticus and non-V. vulnificus strains. By observation, positive results could be obtained only when genomic DNAs of V. parahaemolyticus strains and V. vulnificus strains were used as templates in multiplex MERT-LAMP reactions, and the target pathogens were correctly identified (Figure 7). However, for the reaction tubes of non-V. parahaemolyticus stains, non-V. vulnificus strains and negative control,

Applicability of the MERT-LAMP Technology
In order to evaluate the practicability of MERT-LAMP approach as a surveillance tool for V. parahaemolyticus and V. vulnificus in seafood, the MERT-LAMP methodology was examined by artificially adding V. parahaemolyticus and V. vulnificus strains into oysters. The LoD of MERT-LAMP assay was 92 CFU/reaction of V. parahaemolyticus ICDC-NVP001 and 83 CFU/reaction of V. vulnificus ATCC 27562 in spiked oyster samples without enrichment, and the two target pathogens could be simultaneously detected in a single MERT-LAMP reaction ( Figure 8, Table 2). The non-contaminated oyster sample was found to be negative.

Applicability of the MERT-LAMP Technology
In order to evaluate the practicability of MERT-LAMP approach as a surveillance tool for V. parahaemolyticus and V. vulnificus in seafood, the MERT-LAMP methodology was examined by artificially adding V. parahaemolyticus and V. vulnificus strains into oysters. The LoD of MERT-LAMP assay was 92 CFU/reaction of V. parahaemolyticus ICDC-NVP001 and 83 CFU/reaction of V. vulnificus ATCC 27562 in spiked oyster samples without enrichment, and the two target pathogens could be simultaneously detected in a single MERT-LAMP reaction ( Figure 8, Table 2). The non-contaminated oyster sample was found to be negative.

Applicability of the MERT-LAMP Technology
In order to evaluate the practicability of MERT-LAMP approach as a surveillance tool for V. parahaemolyticus and V. vulnificus in seafood, the MERT-LAMP methodology was examined by artificially adding V. parahaemolyticus and V. vulnificus strains into oysters. The LoD of MERT-LAMP assay was 92 CFU/reaction of V. parahaemolyticus ICDC-NVP001 and 83 CFU/reaction of V. vulnificus ATCC 27562 in spiked oyster samples without enrichment, and the two target pathogens could be simultaneously detected in a single MERT-LAMP reaction ( Figure 8, Table 2). The non-contaminated oyster sample was found to be negative.  The LoD of multiplex MERT-LAMP approach was in conformity with that of conventional LAMP assay only for V. parahaemolyticus or V. vulnificus, respectively (Table 2). Comparatively, the PCR and qPCR methods generated positive results when the contaminate numbers of V. parahaemolyticus ICDC-NVP001 amounted to more than 9200 and 920 CFU/reaction, V. vulnificus ATCC 27562 for 8300 and 830 CFU/reaction, respectively. The results suggested that the sensitivity of multiplex MERT-LAMP approaches was 100-and 10-fold more sensitive than that of PCR and qPCR technologies ( Table 2).

Discussion
V. parahaemolyticus and V. vulnificus, as serious bacterial pathogens for aquatic animals and humans, are widely distributed in coastal and estuarine environments throughout the world [15]. In aquatic animals, the two pathogens are capable of causing serve illnesses in penaeid shrimp, fish, and shellfish, leading to significant losses in aquaculture industries [21]. In humans, most V. parahaemolyticus and V. vulnificus infections resulted from the consumption of raw, inadequately or improperly cooked seafood, particularly shellfish (such as oysters) [1]. Therefore, there is a need for rapid, specific, sensitive, and cost-effective diagnosis methodology that can be used effectively for simultaneous detection of V. parahaemolyticus and V. vulnificus in various samples.
In this report, the first MERT-LAMP assay targeting the toxR gene of V. parahaemolyticus and the rpoS gene of V. vulnificus has was successfully established. The new MERT-LAMP technique could simultaneously detect and distinguish toxR and rpoS genes in only one isothermal step, eliminating the use of temperature-regulating apparatus. Moreover, the MERT-LAMP did not require further processing, and the reaction tubes were not opened in the course of the experiment, which effectively alleviated any carryover contamination. The optimal MERT-LAMP amplification temperature was 60˝C to 64˝C, and 62˝C was selected as reaction temperature. The MERT-LAMP reactions were completed within 60 min. The detection speed of MERT-LAMP method was similar with traditional LAMP assays, while the MERT-LAMP methodology achieved multiplex detection in a single reaction; thus, V. parahaemolyticus and V. vulnificus strains in the same samples could be simultaneously detected and correctly identified (Figures 6 and 8).
Sensitivity of the MERT-LAMP assay was examined using serial dilution of genomic DNA templates. First, we evaluated the MERT-LAMP technique in a single target format by using separate detection of toxR (V. parahaemolyticus-specific gene) and rpoS (V. vulnificus-specific gene) from an V. parahaemolyticus and V. vulnificus DNA templates in each reaction, and the LoD of MERT-LAMP methodology for independently amplifying toxR gene or rpoS gene was 250 fg or 125 fg of genomic DNA templates per reaction, respectively ( Figure 5). The sensitivity of MERT-LAMP method for detecting a single target was consistent with conventional LAMP assay (Figures 2 and 5 Table 1). Second, we extended the MERT-LAMP approach to simultaneously amplify toxR and rpoS genes in a single reaction, and the two target pathogens were successfully identified and detected. The LoD of MERT-LAMP approach for simultaneously detecting toxR and rpoS genes was 250 fg and 125 fg of genomic DNA templates per tube, respectively, and which is the same as that of singleplex MERT-LAMP detections (Figures 5 and 6). The LoD of multiplex MERT-LAMP technology was identical with that the conventional LAMP technique described in this study or developed in other publications, and 10-fold and 100-fold more sensitive than conventional qPCR and PCR techniques (Figures 2 and 6 Table 1) [19]. In addition, the multiplex MERT-LAMP assay was also successfully examined using artificially contaminated oyster samples, and the multiplex MERT-LAMP methodology performed better than PCR and qPCR techniques with respect to detection speed and LoD ( Figure 8 and Table 2).
In addition to its sufficient sensitivity, the MERT-LAMP assay in identification of V. parahaemolyticus and V. vulnificus offered a high degree of specificity and the assay specificity was successfully ascertained. Each primer set contained six specific primers that recognized eight distinct regions on the toxR gene of V. parahaemolyticus and rpoS gene of V. vulnificus, which ensured high specificity of the DNA template detection [7,9,19]. In order to verify the specificity of multiplex MERT-LAMP for detection of V. parahaemolyticus and V. vulnificus, a variety of bacterial strains listed in Table 3 were determined in this study. The results obtained from our study showed that the MERT-LAMP technology indicated a high degree of specificity to V. parahaemolyticus and V. vulnificus by generating positive results to all examined isolates of the two bacteria while producing a negative result to non-V. parahaemolyticus and non-V. vulnificus bacteria strains. Moreover, the two target pathogens were correctly differentiated (Figure 7). These results suggested that the MERT-LAMP assay established here was specific to target sequence detection.
In conclusion, our study is the first report of the use of MERT-LAMP assay for simultaneous detection of V. parahaemolyticus and V. vulnificus, comparing with regular LAMP detection approaches, which provides advantages on real time and multiplex detection. Compared with PCR-based techniques, the MERT-LAMP method is advantageous on sensitivity, specificity, rapidity, time consumption, and ease in operation. Therefore, the developed MERT-LAMP assay can be used as a valuable tool for detection of V. parahaemolyticus and V. vulnificus in basic and field laboratories.

Design of the MERT-LAMP Primers
Based on the toxR gene of V. parahaemolyticus and rpoS gene of V. vulnificus, two sets of MERT-LAMP primers were designed by PrimerExplorer V4 (Eiken Chemical) according to the mechanism of MERT-LAMP methodology [7,9]. Blast analysis validated that two sets of MERT-LAMP primers were specific for V. parahaemolyticus and V. vulnificus. The dark quenchers used were Black Hole Quencher-1 and Black Hole Quencher-2, and the fluorophores used were HEX and Cy5, which are assigned to toxR and rpoS primer sets, respectively. All oligonucleotides described here were synthesized and purified by Tianyi Biotech (Beijing, China). The details of target sequences, primer design, primers sequences and locations were shown in Table 3 and Figure 9. examined using artificially contaminated oyster samples, and the multiplex MERT-LAMP methodology performed better than PCR and qPCR techniques with respect to detection speed and LoD ( Figure 8 and Table 2). In addition to its sufficient sensitivity, the MERT-LAMP assay in identification of V. parahaemolyticus and V. vulnificus offered a high degree of specificity and the assay specificity was successfully ascertained. Each primer set contained six specific primers that recognized eight distinct regions on the toxR gene of V. parahaemolyticus and rpoS gene of V. vulnificus, which ensured high specificity of the DNA template detection [7,9,19]. In order to verify the specificity of multiplex MERT-LAMP for detection of V. parahaemolyticus and V. vulnificus, a variety of bacterial strains listed in Table 3 were determined in this study. The results obtained from our study showed that the MERT-LAMP technology indicated a high degree of specificity to V. parahaemolyticus and V. vulnificus by generating positive results to all examined isolates of the two bacteria while producing a negative result to non-V. parahaemolyticus and non-V. vulnificus bacteria strains. Moreover, the two target pathogens were correctly differentiated (Figure 7). These results suggested that the MERT-LAMP assay established here was specific to target sequence detection.
In conclusion, our study is the first report of the use of MERT-LAMP assay for simultaneous detection of V. parahaemolyticus and V. vulnificus, comparing with regular LAMP detection approaches, which provides advantages on real time and multiplex detection. Compared with PCR-based techniques, the MERT-LAMP method is advantageous on sensitivity, specificity, rapidity, time consumption, and ease in operation. Therefore, the developed MERT-LAMP assay can be used as a valuable tool for detection of V. parahaemolyticus and V. vulnificus in basic and field laboratories.

Design of the MERT-LAMP Primers
Based on the toxR gene of V. parahaemolyticus and rpoS gene of V. vulnificus, two sets of MERT-LAMP primers were designed by PrimerExplorer V4 (Eiken Chemical) according to the mechanism of MERT-LAMP methodology [7,9]. Blast analysis validated that two sets of MERT-LAMP primers were specific for V. parahaemolyticus and V. vulnificus. The dark quenchers used were Black Hole Quencher-1 and Black Hole Quencher-2, and the fluorophores used were HEX and Cy5, which are assigned to toxR and rpoS primer sets, respectively. All oligonucleotides described here were synthesized and purified by Tianyi Biotech (Beijing, China). The details of target sequences, primer design, primers sequences and locations were shown in Table 3 and Figure 9.

Reagents
Loopamp TM Fluorescent Detection Reagent (FD) and the Loopamp kits were purchase from Eiken Chemical (Tokyo, Japan, and Beijing, China). The DNA extraction kits (QIAamp DNA Mini Kits) were purchased from Qiagen (Beijing, China), and the Nb.BsrDI was purchased from New England Biolabs (Beijing, China).

Bacterial Strains
The bacterial strains tested in this study were listed in Table 4. All Vibrio strains were routinely cultured using thiosulfate citrate bile salt sucrose agar (TCBS agar, Eiken Chemical) at 35˝C overnight. Storage and culture conditions of non-Vibrio strains were followed as previously described [11]. The extracted templates were tested with ultraviolet spectrophotometer at A260/280 and stored under at´20˝C until use.

The Conventional LAMP Assay
In order to examine the availability of two LAMP primer sets, the LAMP amplification either for V. parahaemolyticus strains or V. vulnificus strains was carried out as the following description. Briefly, the traditional LAMP reaction was performed with the Loopamp Kit in a final volume of 25 µL containing 1.6 µM each FIP and BIP primers, 0.8 µM each LF and LB primers, 0.4 µM each F3 and B3 primers, 12.5 µL 2ˆreaction mix, 1 µL of Bst DNA polymerase (8 U), 1 µL FD and 1 µL DNA template.
The amplification mixtures of convention LAMP were heated at 62˝C for 1 h then at 85˝C for 5 min to stop the amplification. Three methods were employed to monitor the conventional LAMP reaction. First, a real-time turbidimeter (LA-320C, Eiken Chemical Co., Ltd., Tokyo, Japan) was used to minor the LAMP reaction by recording the optical density (OD) at 650 nm every 6 s. The turbidity readings were produced in real-time format and a turbidity threshold value of 0.1 was defined. Second, the positive reactions could be directly seen color change by FD reagent. Moreover, the amplification products were also detected by electrophoresis on 2% agarose gels with ethidium bromide staining. Mixture without DNA template was used as a negative control.
A real-time machine (Rotor-Gene Q, Qiagen, Hilden, Germany) was applied to run the MERT-LAMP reactions, and the assay was conducted at 62˝C for 1 h. Mixture without DNA template was used as a negative control. After amplification, the MERT-LAMP products were confirmed by electrophoresis on 2% agarose gels with ethidium bromide staining or directly observed the color change by FD reagent.
To assess the optimal reaction temperature of MERT-LAMP approach, we mixed the MERT-LAMP reaction mixtures at a constant temperature ranging from 60˝C to 67˝C for 60 min and then heated at 85˝C for 5 min to complete the reaction. Mixtures without DNA template were used as a negative control.

Determination of the Sensitivity of the MERT-LAMP in Pure Cultures
Strains of V. parahaemolyticus ICDC-NVP001 and V. vulnificus ATCC 27562 were used for the assay of sensitivity testing with pure culture, and the genomic DNA templates were serially diluted. Individual MERT-LAMP approaches, which target the toxR and rpoS genes individually, were carried out with serial dilutions (2.5 ng, 250 pg, 25 pg, 2.5 pg, 250 fg, 125 fg, 62.5 fg, and 31.25 fg per microliter) according to standard MERT-LAMP system. To make a comparative analysis of MERT-LAMP, LAMP, qPCR and PCR assays by using pure culture, the limit of detection (LoD) of LAMP, qPCR, and PCR assays was defined by genomic DNA amount of the template. The V. parahaemolyticus-qPCR, V. parahaemolyticus-PCR, V. vulnificus-qPCR, and V. vulnificus-PCR assays have been established in previous studies, which were selected for confirming the LoD of qPCR and PCR technologies [22][23][24][25].
To test the sensitivity of multiplex MERT-LAMP assay, the MERT-LAMP reaction was conducted under the conditions described above with serial dilutions (2.5 ng, 250 pg, 25 pg, 2.5 pg, 250 fg, 125 fg, 62.5 fg, and 31.25 fg per microliter) of V. parahaemolyticus and V. vulnificus, and 1µl DNA template DNA each of V. parahaemolyticus strains and V. vulnificus strains was simultaneously added into a MERT-LMAP reaction.

Evaluation of the Specificity of the MERT-LAMP Assay
To assess the MERT-LAMP technology's specificity, the multiplex MERT-LAMP reactions were carried out under the conditions described above with the purely genomic DNA templates extracted from 111 strains ( Table 2). Analysis of each sample was performed twice independently.

Practical Application of MERT-LAMP to V. parahaemolyticus and V. vulnificus Detection in Oyster Samples
In order to determine practicability of MERT-LAMP approach in identification of V. parahaemolyticus and V. vulnificus, V. parahaemolyticus (ICDC-NVP001), and V. vulnificus (ATCC27562) were simultaneously added to oyster samples, which were purchased at a local market in Beijing. The oyster samples were confirmed as being V. parahaemolyticus and V. vulnificus negative by traditional culture assay and PCR [23,25]. Only oyster samples, which were negative for V. parahaemolyticus and V. vulnificus, were applied to spiked oyster samples.
Firstly, to test the minimal detectable colony forming units (CFUs), the cultures with V. parahaemolyticus and V. vulnificus strains were serially diluted (10´1 to 10´9), and the aliquots of 100 µL appropriate dilution (10´6) was plated in triplicate on brain heart infusion (BHI). The CFUs were counted after 24 h at 37˝C [20]. The following procedures, one hundred microliters of appropriate dilutions of V. parahaemolyticus and V. vulnificus with known amounts was simultaneously spiked into 800 µL of each of the oyster homogenates and mixed well. The oyster homogenates was centrifuged at 200 g for 5 min and the supernatant was transferred to a new vessel, and then was centrifuged at 18,000 g for 5 min. After removal of the supernatant, the pellet was applied for DNA extraction. For MERT-LAMP, LAMP, real-time PCR, and PCR assays, 1 µL of each supernatants (2 µL) were used as templates. Non-contaminated oyster sample was chosen as negative control. This performance was carried out in triplicate independently.