Rabbit anti-fluorescein antibody (anti-FITC Ab) and biotinylated bovine serum albumin (biotin-BSA) were purchased from the Abcam Co., Ltd. (Shanghai, China). Streptavidin-immobilized 30-nm gold nanoparticles (SA-G) was purchased from the Resenbio Co., Ltd. (XiAn, China). Membrane backing materials, sample and conjugate pads, nitrocellulose membrane (NC), and absorbent pads were purchased from the Jie Yi Biotechnology Co., Ltd. (Shanghai, China). The Loopamp kits were purchased from Eiken Chemical Co., Ltd. (Beijing, China). QIAamp DNA Mini Kit (QIAamp DNA minikits; Qiagen, Hilden, Germany) was purchased from Qiagen Co., Ltd. (Beijing, China). The visual detection reagent (Hydroxynaphthol blue, HNB) was purchased from BeiJing-HaiTaiZhengYuan Technology Co., Ltd. (Beijing, China).

The dry-reagent strips (4 mm × 60 mm) were prepared as previously described with some modifications (Wang et al., 2016b,d). In brief, the sample pad, conjugate pad, NC membrane and absorbent pad were laminated onto a plastic adhesive backing card. The anti-FITC Ab (0.15 mg/ml) and biotin-BSA (2.5 mg/ml) conjugates were sprayed onto NC membrane to form the test line (TL) and control line (CL), with each line separated by 5 mm. SA-G in 0.01M PBS (PH 7.4) was deposited on the conjugate pad of the strip. The assembled cards were cut into 4-mm wide strips (Deli No. 8012). The assembled biosensors were packaged in a plastic box containing a desiccant gel and stored at the room temperature.

The strains employed in this study (Table 1) were stored in 10% (w/v) glycerol broth at -70°C. The Vibrio isolates were cultured three times on thiosulfate citrate bile salt sucrose agar (TCBS agar, Eiken Chemical) at 35°C and the non-Vibrio strains were cultured three times on nutrient agar plate at 37°C. Genomic DNA was extracted from all culture strains using the QIAamp DNA Mini Kit according to the manufacturer’s instructions and quantified using a Nano drop ND-1000 instrument (Calibre, Beijing, China). V. parahaemolyticus ICDC-NVP001 was serially diluted (10 ng, 10 pg, 10 fg, 1 fg, and 0.1 fg) for sensitivity analysis of V. parahaemolyticus-MCDA-LFB detection.

The MCDA primer pairs (F1, F2, CP1, CP2, C1, C2, D1, D2, R1 and R2) were designed using PrimerExplorer V4 (Eiken Chemical, Japan) and primer software PRIMER PREMIER 5.0. All primers were analyzed for hairpin structures and hybrids using the Integrated DNA Technologies design tools¹. Blast analysis was used to verify that the MCDA primers were specific for V. parahaemolyticus. The CP1 (C1+P1) and C1 primers were labeled at their 5′ end with biotin and fluorescein isothiocyanate (FITC), respectively. The sequences, positions and modifications of the primer pairs are displayed in Figure 1 and Table 2. All of the oligomers were synthesized and purified by TsingKe Biotech Co., Ltd. (Beijing, China) at HPLC purification grade.

Multiple cross displacement amplification reactions were carried out in 25 μl amplification mixtures as previous studies (Wang Y. et al., 2015; Wang et al., 2016b,c,d). Briefly, each reaction contained 0.4 μM each of displacement primers F1 and F2, 0.8 μM each of amplification primers C1^∗ and C2, 1.2 μM each of amplification primers R1, R2, D1 and D2, 1.2 μM each of cross primers CP1 and CP1^∗, 2.4 μM cross primer CP2, 12.5 μl 2× reaction mix (Loopamp DNA amplification Kit), 1.25 μl of Bst DNA polymerase (10 U) and 1 μl DNA template.

A total of four monitoring methods, including colorimetric indicator (HNB), gel electrophoresis, turbidimeter (LA-320C) and LFB detection, were used for analyzing the amplicons. When employing HNB, the amplified products caused a color change from violet to sky blue, while the negative controls and blank control remained violet. MCDA products were analyzed by 2% agarose gel electrophoresis, the specific ladder of multiple bands should be seen for positive amplifications, but not in the negative and blank controls. By LFB, two visible red lines (TL; CL) should be observed in positive reactions, and only the control lines were visual in negative and blank controls.

The optimal reaction temperature was determined in the range of 60 to 67°C for 60 min. Mixtures with 1 μl genomic template of Enterococcus faecalis strains (E. faecalis, ATCC35667) and Shigella flexneri (S. flexneri, ICDC-NPS001) strains were used as negative controls, and mixtures with 1 μl double distilled water (DW) were selected as a blank control.

The specificity of V. parahaemolyticus-MCDA-LFB was analyzed with DNA templates from 143 bacterial strains (Table 1). The assays were repeated at least twice. The limit of detection (LoD), which was tested using serial dilutions (10 ng, 10 pg, 10 fg, 1 fg and 0.1 fg per microliter), was defined by genomic DNA amount of the template. Detection by V. parahaemolyticus-MCDA-LFB was compared to that with a colorimetric indicator (HNB), real time turbidity and 2% agarose gel electrophoresis. Three replicates of each dilution were tested.

The optimal time for V. parahaemolyticus-MCDA-LFB was determined by increasing the reaction time from 10 to 40 min at 10 min intervals. The MCDA products were analyzed using LFB detection and two replicates of each amplification time were determined.

Vibrio parahaemolyticus ICDC-NVP001 was added into oyster samples obtained from local seafood restaurants in Beijing. Only oyster samples that tested negative for V. parahaemolyticus according to Kim et al. (1999) were spiked. V. parahaemolyticus cultures were serially diluted (10^-1 to 10^-8), and 100-μl aliquots (appropriate dilution:10^-6) were placed in triplicate on brain heart infusion (BHI). CFUs were counted after 24 h at 37°C. Simultaneously, 0.1 mL of diluted V. parahaemolyticus cultures (10^-3 to 10⁸) with known amounts (4.2 × 10⁵ to 4.2 × 10⁰ CFU/mL) was inoculated into 900 μl of oyster homogenates and mixed well. The spiked oyster samples were centrifuged at 200 g for 5 min, and the supernatant was placed into a new tube, and the was centrifuged at 18000 g for 5 min. The supernatant was removed and the pellet was subjected to extract genomic DNA, and the DNA templates were eluted in 20 μl of elution buffer. For the MCDA-LFB assay, 1 μl of the extracted DNA was used as template and non-contaminated oyster samples were served as negative control. Three independent assays were performed.

To determine the availability of V. parahaemolyticus-MCDA primers (Table 2), V. parahaemolyticus MCDA assays with DNA from pure cultures were carried out at 62°C for 1 h. Amplification occurred with DNA from V. parahaemolyticus (ICDC-NVP001), but not with E. faecalis (ATCC 35667), S. flexneri (ICDC-NPS001) DNA and the DW control (Figure 2). Therefore, the V. parahaemolyticus-MCDA primer set was a good candidate for development of the MCDA-LFB assay for V. parahaemolyticus detection.

To verify the optimum amplification temperature, V. parahaemolyticus strain (ICDC-NPV001) was used as the positive control at the level of 10 pg per reaction and the reactions were monitored by the real time turbidity method. Carrying out MCDA at temperatures from 60 to 67°C at 1°C increments confirmed that 62°C was the most suitable temperature for amplification as indicated by the kinetics graphs displayed in Figure 3. At all tested temperatures, the typical kinetics graphs were generated, with the faster amplification obtained from assay temperatures of 62°C.

When DNA from the bacteria listed in table 1 was used in MCDA-LFB assays, only the DNA from the V. parahaemolyticus strains provided results. DNA from other Vibrio species and from all non-Vibrio isolates did not lead to the production of detectable amplification products (Figure 4). Two red lines, including TL and CL, appeared on the strips for the positive tests, and only a red line (CL) appeared on the biosensors, suggesting negative results for non-V. parahaemolyticus isolates and blank control.

Serial dilution of the V. parahaemolyticus genomic DNA in triplicate and use in MCDA assays demonstrated that as little as 10 fg of template DNA produced sufficient amplified DNA for detection by the four monitoring methods employed (Figure 5). The results obtained using biosensor were in complete accordance with real time turbidity, HNB reagent and agarose gel electrophoresis analysis (Figure 5). Moreover, it was possible to detect the amplicons when the amplification reaction was carried out for 30 min (Figure 6).

The lowest number of V. parahaemolyticus CFUs could be detected in 1 mL of spiked oyster homogenate was approximately 4.2 × 10² CFU/mL (∼2.1 CFU per reaction; Figure 7). The V. parahaemolyticus-MCDA assay produced negative results at the concentrations lower than 4.2 × 10¹ CFU/mL (∼0.21 CFU per reaction), negative control and blank control. As seen with MCDA assays utilizing DNA from pure cultures, detection of the amplicons was as sensitive with the LFB method with the other three methods (Figure 7).