| Vibrio harveyi, Vibrio parahaemolyticus, Vibrio scophthalmi, Vibrio anguillarum, and Photobacterium damselae subsp. damselae are important pathogenic bacteria frequently reported in mariculture animal diseases in recent years. These pathogens exhibit strong pathogenicity, wide epidemic area, and high mortality rates, which usually cause serious economic losses in aquaculture. Strengthening the research on rapid detection technology of these pathogens can help to effectively prevent and control their transmission and infection, and reduce economic losses of the aquaculture industry. Therefore, rapid detection of multiple pathogens in this field can promote the disease control technology development of the aquaculture industry. In this study, the vhhA gene of V. harveyi, toxR gene of V. parahaemolyticus, luxR gene of V. scophthalmi, empA gene of V. anguillarum, and Mcp gene of P. damselae subsp. damselae were selected as the target genes. Specific primers were designed using Primer Premier 5.0 software. V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae were used as the target bacteria, V. campbelii and 10 other bacterial species were used as the control group, and sterile water was used as the blank control. The specificity of the primers was verified by the laboratorial conventional real-time quantitative PCR. Thereafter, V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae were amplified using the UF-150 microfluidic fluorescence quantitative PCR instrument to verify the feasibility and specificity of the designed primers on microfluidic fluorescence quantitative PCR. The PCR products were digested and recycled, then ligated to PMD-19T vector and transformed into DH-5α competent cells to construct the standard reference material. The extracted plasmid DNA was used as a template for fluorescence quantitative PCR amplification after 10-fold gradient dilution, and a standard curve was drawn. The DNA of V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae were used to optimize the annealing temperatures for single microfluidics quantitative PCR. The annealing temperatures were set at 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, and 63 ℃, respectively, and the optimal common annealing temperatures of the five strains were finally screened. Meanwhile, the number of reaction cycles was set at 25, 30, 35 and 40, respectively, to verify the optimal number of reaction cycles. The designed specific primers of V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae were integrated into the microfluidic chip; multiple microfluidic fluorescence quantitative PCR was performed on the integrated chip with the DNA template of the five strains to verify the cross-reaction between the primers. Based on the reaction systems and conditions of single detection methods, the reaction systems and conditions of multiple microfluidic fluorescence quantitative PCR were optimized, and the microfluidic chip was integrated to establish a multiple microfluidic fluorescence quantitative PCR detection technology that could simultaneously detect these five pathogens: V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum and P. damselae subsp. damselae. Scophthalmus maximus, Litopenaeus vannamei, and Apostichopus japonicus were selected as the experimental orgainsms. The five pathogens were mixed with the tissues of S. maximus (muscle, gill and liver), L. vannamei (muscle and gill), and A. japonicus (respiratory tree and intestine). The established multiple microfluidic fluorescence quantitative PCR method was used to detect these tissues which contained the five pathogens, and the conventional real-time fluorescence quantitative PCR was performed for comparison. The results showed that the optimal reaction system of the established detection method was as follows: 2×Taq Pro Universal SYBR qPCR Master Mix 5 μL, primer F/R 1 μL, DNA template 2 μL, and ddH2O 1 μL. The reaction conditions were as follows: 95 ℃ for 30 s, 95 ℃ for 5 s, 61 ℃ for 30 s, and 30 cycles of amplification. The standard curves of V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae showed good linearity in the range of 104–109 copies/μL. The linear equations were as follows: y= ‒2.972x+6.73, R2= 0.999; y= ‒3.287x+7.48, R2=0.998; y= ‒3.549x+8.14, R2=0.998; y= ‒3.912x+7.83, R2=0.999; y= ‒3.969x+7.07, R2=0.992, respectively. The method showed strong specificity and high sensitivity to V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae, and the minimum detection limits for these bacteria were 40, 200, 200, 500 and 20 CFU/mL, respectively. The established multiplex microfluidic fluorescence quantitative PCR method was used to detect these pathogens in animal samples and compare the results to those of conventional fluorescence quantitative PCR. The results showed that the accuracy of the established multi-microfluidic fluorescence quantitative PCR method was higher than 96.2% of the conventional real-time fluorescence quantitative PCR method. The average detection time of the samples was only 26 min, which was significantly shorter than that of the conventional real-time fluorescence quantitative PCR reaction time of 1 h 40 min. The multiplex microfluidic fluorescence quantitative PCR method established in this study for the detection of five kinds of mariculture pathogens has strong specificity, high sensitivity, low environmental condition requirements, outstanding portability, and detection accuracy (same as that of conventional real-time fluorescence quantitative PCR), which is suitable for the development of rapid detection technology for aquatic pathogens. |
