渔业科学进展  2023, Vol. 44 Issue (3): 222-234  DOI: 10.19663/j.issn2095-9869.20211224001
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引用本文 

徐媛媛, 于永翔, 王印庚, 王春元, 李永杰, 刘定远, 秦蕾, 张正. 5种主要海水养殖病原菌多重微流控荧光定量PCR快速检测技术的建立[J]. 渔业科学进展, 2023, 44(3): 222-234. DOI: 10.19663/j.issn2095-9869.20211224001.
XU Yuanyuan, YU Yongxiang, WANG Yingeng, WANG Chunyuan, LI Yongjie, LIU Dingyuan, QIN Lei, ZHANG Zheng. Establishment of Multiple Microfluidic Fluorescence Quantitative PCR Detection Technology for Five Main Mariculture Bacterial Pathogens[J]. Progress in Fishery Sciences, 2023, 44(3): 222-234. DOI: 10.19663/j.issn2095-9869.20211224001.

基金项目

国家重点研发计划课题(2019YFD0900104)、山东省自然科学基金项目(ZR2020QC216; ZR2021MC027)和江苏省研究生科研与实践计划项目(SJCX20_1288)共同资助

作者简介

徐媛媛，E-mail: 759645785@qq.com

通讯作者

张正，副研究员，E-mail: zhangzheng@ysfri.ac.cn

文章历史

收稿日期：2021-12-24
收修改稿日期：2022-02-14
Contents              Abstract              Full text              Figures/Tables              PDF
5种主要海水养殖病原菌多重微流控荧光定量PCR快速检测技术的建立
徐媛媛 1,2, 于永翔 2, 王印庚 2,3, 王春元 2, 李永杰 2, 刘定远 1,2, 秦蕾 1, 张正 2,3     
1. 江苏海洋大学 江苏 连云港 222005;
2. 农业农村部海水养殖病害防治重点实验室 中国水产科学研究院黄海水产研究所 山东 青岛 266071;
3. 青岛海洋科学与技术试点国家实验室海洋渔业科学与食品产出过程功能实验室 山东 青岛 266071
收稿日期：2021-12-24；收修改稿日期：2022-02-14
基金项目：国家重点研发计划课题(2019YFD0900104)、山东省自然科学基金项目(ZR2020QC216; ZR2021MC027)和江苏省研究生科研与实践计划项目(SJCX20_1288)共同资助
作者简介：徐媛媛，E-mail: 759645785@qq.com.
通讯作者：张正，副研究员，E-mail: zhangzheng@ysfri.ac.cn.
摘要：在养殖现场开展多病原的快速检测是水产养殖产业健康发展的重要技术需求之一。本研究选取哈维氏弧菌(Vibrio harveyi)的vhhA基因、副溶血弧菌(Vibrio parahaemolyticus)的toxR基因、大菱鲆弧菌(Vibrio scophthalmi)的luxR基因、鳗弧菌(Vibrio anguillarum)的empA基因和美人鱼发光杆菌美人鱼亚种(Photobacterium damselae subsp. damselae)的Mcp基因作为靶基因，设计特异性引物，通过优化反应体系和条件，并在微流控芯片进行集成，建立了可同步检测这5种病原菌的多重微流控荧光定量PCR检测技术。结果显示，本研究所建立的检测技术最佳反应体系：2×Taq Pro Universal SYBR qPCR Master Mix 5 μL，Primer F/R各1 μL，DNA模板2 μL，ddH2O 1 μL。反应条件为95 ℃ 30 s，95 ℃ 5 s，61 ℃ 30 s，扩增30个循环。通过绘制标准曲线发现，在109~104 copies/μL浓度范围内均有良好的线性相关性。该方法对哈维氏弧菌、副溶血弧菌、大菱鲆弧菌、鳗弧菌和美人鱼发光杆菌美人鱼亚种特异性强，对5种病原菌的最低检测限分别为40、20、200、500和20 CFU/mL，显示出较高的灵敏度，且样品平均检测时间缩短至26 min左右。本研究结果为开发水产养殖多病原快速、精准的现场检测技术奠定了重要基础。
关键词：哈维氏弧菌    副溶血弧菌    大菱鲆弧菌    鳗弧菌    美人鱼发光杆菌美人鱼亚种    微流控PCR    现场快速检测    
Establishment of Multiple Microfluidic Fluorescence Quantitative PCR Detection Technology for Five Main Mariculture Bacterial Pathogens
XU Yuanyuan 1,2, YU Yongxiang 2, WANG Yingeng 2,3, WANG Chunyuan 2, LI Yongjie 2, LIU Dingyuan 1,2, QIN Lei 1, ZHANG Zheng 2,3     
1. Jiangsu Ocean University, Lianyungang 222005, China;
2. Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academic of Fishery Sciences, Qingdao 266071, China;
3. Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266071, China
Corresponding author: ZHANG Zheng, E-mail: zhangzheng@ysfri.ac.cn.
Abstract: 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.
Key words: Vibrio harveyi    Vibrio parahaemolyticus    Vibrio scophthalmi    Vibrio anguillarum    Photobacterium damselae subsp. damselae    Microfluidic fluorescence quantitative PCR    On-site rapid detection    

进入21世纪以来，我国的海水养殖产业发展迅猛，养殖规模不断扩大，产量不断增加，但随之而来的养殖密度过高、水环境恶化等诸多问题导致病害频发，给产业造成了严重的经济损失。其中，细菌性疾病成为海水养殖中最常见的问题之一，哈维氏弧菌(Vibrio harveyi)、副溶血弧菌(Vibrio parahaemolyticus)、大菱鲆弧菌(Vibrio scophthalmi)、鳗弧菌(Vibrio anguillarum)和美人鱼发光杆菌美人鱼亚种(Photobacterium damselae subsp. damselae)是近年来频繁见于文献报道的几种重要的海水养殖动物致病菌，可感染海水鱼类(张飞等, 2012; 陈吉祥等, 2005)、对虾(陈月忠等, 2000; 李翠苹等, 2020)和贝类(Hidalgo et al, 2008)等多种海水经济动物发病。这些病原菌具有致病性强、流行面积广、致死率高等显著特点，对水产养殖生产危害很大，加强对这些病原菌的快速检测技术研究，有助于有效防控其传播和感染，减少养殖业的经济损失。

荧光定量PCR技术因在检测通量、精准性、灵敏度以及成本方面的突出优势而得到迅速发展(何军霞等, 2021)，被广泛应用于食品安全(雷庆等, 2021)、医学诊断(Fidalgo et al, 2021)和畜牧兽医(张若楠等, 2020)等众多领域。微流控技术(microfluidics)又被称为芯片实验室，是一种以在微米尺度空间对流体进行操控为主要特征的科学技术(陈京等, 2020)。将微流控技术与荧光定量PCR技术相结合，形成的微流控荧光定量芯片检测是利用微流控器件热质量小和比面积高等特点实现快速传热，从而缩短PCR扩增过程中稳定温度需要的时间，与常规的实验室荧光定量PCR仪器相比，微流控荧光定量PCR技术具有检测所需样品量少、升降温速度快、耗时短、成本低、便于携带、环境条件要求低等显著特点，在疾病临床诊断(王可可等, 2018)、食品检测(吴姗等, 2009)和畜牧兽医(王楷宬等, 2021)等多个领域已经得到广泛应用。

本研究基于微流控荧光定量PCR技术，针对哈维氏弧菌、副溶血弧菌、大菱鲆弧菌、鳗弧菌和美人鱼发光杆菌美人鱼亚种设计特异性引物，通过优化反应体系和反应条件，进行这5种病原菌在微流控检测芯片的集成应用，实现重要海水养殖病原菌的微流控荧光定量PCR一步式快速检测，研究结果将为开发在水产养殖现场应用的病原菌多重微流控检测技术奠定基础。

1 材料与方法 1.1 实验菌株

本研究使用15种细菌，共计28株。其中，灿烂弧菌(Vibrio splendidus)标准菌株购自中国科学院微生物研究所中国普通微生物菌种保藏管理中心(China General Microbiological Culture Collection Center, CGMCC)，坎贝氏弧菌(Vibrio campbelii)等11株标准菌株购自自然资源部第三海洋研究所海洋微生物菌种保藏管理中心(Marine Culture Collection of China, MCCC)，哈维氏弧菌等17株菌株由本实验室分离自不同的患病水产动物。菌株具体信息见表 1。

表 1 实验所用菌株 Tab.1 The strains for testing
1.2 微流控PCR仪器及反应芯片

本研究选用UF-150微流控荧光定量PCR仪(GENECHECKERTM Ultra-Fast Real-time PCR System)，由韩国Genesystem公司生产，其微流控反应芯片由聚甲基丙烯酸甲酯材料制成，尺寸大小为38 mm×25 mm× 6 mm (图 1)。该芯片共有10个反应通道且都标有孔号，奇数孔号在芯片顶部，偶数孔号在芯片底部，加样孔位于孔号一侧，每个加样孔对面是通气孔，反应通道内的空气和加样时的过量液体会通过通气孔排出。每个反应通道的容量为10 μL，将荧光染料预混液、引物和待测样品混匀后加入不同反应通道后，用封膜胶带密封每个孔，以防止在热循环期间样品蒸发和污染。反应结束后，可通过查看反应通道的荧光信号强弱和扩增曲线来判定结果。

图 1 微流控芯片结构图与实物 Fig.1 Microfluidic chip structure diagram and product (a)结构图；(b)实物；①、⑤：芯片翼；②：孔号；③：加样孔；④：通气孔。 (a) Structure of the chip; (b) Product of the chip; ①, ⑤: Chip wing; ②: Hole numbers; ③: Loading hole; ④: Vent hole.
1.3 细菌模板DNA的制备

首先将保存于–80 ℃的28株细菌接种于TSB培养基，28 ℃培养18 h活化后，挑取单菌落转接至新的TSB平板培养基，在28 ℃下纯化培养18 h，然后挑取1~2个单菌落于100 μL的无菌水中混匀，100 ℃金属浴15 min，再12 000 r/min离心5 min，收集上清液即为细菌的DNA模板，保存于–20 ℃备用。

1.4 引物设计及特异性验证

根据GenBank中登录的哈维氏弧菌vhhA基因、副溶血弧菌toxR基因和大菱鲆弧菌luxR基因的保守序列，用Primer Premier 5.0软件设计特异性引物，再通过NCBI中Blast验证引物的特异性。鳗弧菌empA基因和美人鱼发光杆菌美人鱼亚种Mcp基因的特异性引物分别参考本实验室已有研究结果(刘智超, 2012; Zhang et al, 2021)。设计好的引物序列交由生工生物工程(上海)股份有限公司合成，引物序列见表 2。

表 2 5株病原菌的特异性引物序列 Tab.2 The specific primer sequences of 5 pathogen strains

以哈维氏弧菌、副溶血弧菌、大菱鲆弧菌、鳗弧菌和美人鱼发光杆菌美人鱼亚种作为检测目标菌，坎贝氏弧菌等其他10种细菌作为对照菌，无菌水作为空白对照，应用实验室常规实时荧光定量PCR仪(Eppendorf AG 22331 Hamburg, 德国)进行扩增，验证各引物的特异性。哈维氏弧菌、副溶血弧菌、大菱鲆弧菌反应体系为2×Taq Pro Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd) 10 μL，Primer F/R各0.4 μL，DNA模板1 μL，ddH2O 8.2 μL。反应条件为95 ℃预变性2 min，95 ℃变性5 s，60 ℃退火30 s，扩增40个循环。鳗弧菌和美人鱼发光杆菌美人鱼亚种反应体系与反应条件参考刘智超(2012)和Zhang等(2021)。

将哈维氏弧菌、副溶血弧菌、大菱鲆弧菌、鳗弧菌和美人鱼发光杆菌美人鱼亚种分别应用UF-150微流控荧光定量PCR仪进行微流控荧光定量PCR扩增，验证引物在微流控仪器上应用的可行性和特异性，反应总体系设定为10 μL：2×Taq Pro Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd) 5 μL，Primer F/R各1 μL，DNA模板2 μL，ddH2O 1 μL。反应条件为95 ℃预变性30 s，95 ℃变性5 s，退火时间30 s，退火温度与普通实时荧光定量PCR一致，扩增30个循环。

1.5 标准曲线的建立

将上述特异性引物分别进行普通PCR扩增，扩增产物切胶回收后连接至PMD-19T载体，转化至DH-5α感受态细胞，增菌培养进行菌液PCR扩增后测序筛选出阳性菌作为标准品。将标准品增菌培养后提取质粒，按10倍梯度稀释6个梯度分别进行荧光定量PCR扩增并绘制标准曲线。

1.6 多病原微流控荧光定量PCR快速检测条件优化

分别用哈维氏弧菌、副溶血弧菌、大菱鲆弧菌、鳗弧菌和美人鱼发光杆菌美人鱼亚种的DNA模板，比较单重微流控荧光定量PCR的退火温度，分别设定反应温度为58、59、60、61、62和63 ℃进行扩增，最终筛选5株菌最佳的共同退火温度。同时分别设反应循环数为25、30、35、40，验证最佳反应循环数。

1.7 集成芯片引物交叉反应分析

将设计好的哈维氏弧菌、副溶血弧菌、大菱鲆弧菌、鳗弧菌和美人鱼发光杆菌美人鱼亚种的特异性引物集成到微流控芯片上，并用制备好的5株菌的DNA模板在集成的芯片上进行多重微流控荧光定量PCR扩增，验证引物间有无交叉反应。反应总体系设定为10 μL：2×Taq Pro Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd) 5 μL，Primer F/R各1 μL，DNA模板2 μL，ddH2O 1 μL。反应条件为95 ℃预变性30 s，95 ℃变性5 s，60 ℃退火30 s，扩增30个循环。

1.8 多病原微流控荧光定量PCR快速检测灵敏度分析

按1.3章节相同的方法，将哈维氏弧菌、副溶血弧菌、大菱鲆弧菌、鳗弧菌和美人鱼发光杆菌美人鱼亚种划线接种于TSB平板培养基进行纯化培养。取适量纯化好的菌苔用无菌PBS溶液分别配制成4.0× 106、2.0×106、2.0×106、5.0×106和2.0×106 CFU/mL浓度的菌悬液，按10倍梯度依次稀释6个梯度，涂布计数以验证其浓度的准确性。将不同浓度梯度的菌悬液按照1.3章节相同的方法制备细菌DNA模板，以灭菌的去离子水为阴性对照，在优化后的反应条件下分别进行多重微流控荧光定量PCR反应，确定该方法的最低检测限。

1.9 多病原微流控荧光定量PCR快速检测技术的应用效果

选取大菱鲆(Scophthalmus maximus)、凡纳滨对虾(Litopenaeus vannamei)、仿刺参(Apostichopus japonicus)为实验对象，分别取大菱鲆的肌肉、鳃、肝组织，凡纳滨对虾的肌肉、鳃组织，仿刺参的呼吸树、肠道组织各0.1 g，分别加入100 μL哈维氏弧菌(1.0× 106 CFU/mL)、副溶血弧菌(1.2×106 CFU/mL)、大菱鲆弧菌(1.6×106 CFU/mL)、鳗弧菌(2.3×106 CFU/mL)和美人鱼发光杆菌美人鱼亚种(8.0×106 CFU/mL)的菌液，同时，以未加菌液的组织作为对照组，将组织研磨匀浆后取10 μL加入100 μL裂解液(Vazyme Biotech Co., Ltd)混匀，100 ℃金属浴15 min后加入100 μL稳定液(Vazyme Biotech Co., Ltd)混匀，12 000 r/min离心5 min，取上清液作为模板，在已经优化好的反应条件下进行多重微流控荧光定量PCR扩增，验证检测效果。

2 结果 2.1 引物特异性验证

应用所设计的引物分别进行普通荧光定量PCR和微流控荧光定量PCR反应，引物特异性验证的结果显示，哈维氏弧菌、副溶血弧菌、大菱鲆弧菌、鳗弧菌和美人鱼发光杆菌美人鱼亚种DNA出现特异性扩增曲线，其他细菌DNA及阴性对照无扩增曲线出现(图 2)，表明所设计的5种菌的引物特异性良好。

图 2 引物特异性验证 Fig.2 Primer specificity validation a：普通实时荧光定量PCR特异性实验；b：微流控荧光定量PCR特异性实验；Ⅰ：哈维氏弧菌；Ⅱ：副溶血弧菌；Ⅲ：大菱鲆弧菌；Ⅳ：鳗弧菌；Ⅴ：美人鱼发光杆菌美人鱼亚种。 a: Conventional real-time quantitative PCR specificity test; b: Microfluidic fluorescence quantitative PCR specific test; Ⅰ: V. harveyi; Ⅱ: V. parahaemolyticus; Ⅲ: V. scophthalmi; Ⅳ: V. anguillarum; Ⅴ: P. damselae subsp. damselae.
2.2 多病原微流控荧光定量PCR快速检测体系集成与反应条件优化

在单重微流控荧光定量PCR反应条件的基础上，对多重微流控荧光定量PCR方法的反应条件进行优化。最佳反应条件下的反应结果如图 3所示，最终确定多重微流控荧光定量PCR反应体系为2×Taq Pro Universal SYBR qPCR Master Mix 5 μL，Primer F/R各1 μL，DNA模板2 μL，ddH2O 1 μL。反应条件为95 ℃ 30 s，95 ℃ 5 s，61 ℃ 30 s，扩增30个循环。

图 3 退火温度61 ℃时微流控荧光定量PCR反应结果 Fig.3 Microfluidic fluorescence quantitative PCR result at annealing temperature 61 ℃ 1、6：哈维氏弧菌引物；2、7：鳗弧菌引物；3、8：美人鱼发光杆菌美人鱼亚种引物；4、9：副溶血弧菌引物；5、10：大菱鲆弧菌引物；6~10：空白对照；a：哈维氏弧菌；b：鳗弧菌；c：美人鱼发光杆菌美人鱼亚种；d：副溶血弧菌；e：大菱鲆弧菌。 1, 6: Primers of V. harveyi; 2, 7: Primers of V. anguillarum; 3, 8: Primers of P. damselae subsp. damselae; 4, 9: Primers of V. parahaemolyticus; 5, 10: Primers of V. scophthalmi; 6~10: Negative control; a: V. harveyi; b: V. anguillarum; c: P. damselae subsp. damselae; d: V. parahaemolyticus; e: V. scophthalmi.
2.3 标准曲线的建立

将哈维氏弧菌、副溶血弧菌、大菱鲆弧菌、鳗弧菌和美人鱼发光杆菌美人鱼亚种的质粒标准品分别按10倍梯度稀释6个梯度，进行荧光定量PCR扩增，并绘制标准曲线。结果如图 4所示，哈维氏弧菌的标准曲线在2.42×104~2.42×109copies/μL浓度范围内有良好的线性相关性(y= –2.972x+6.73, R2=0.999)，副溶血弧菌的标准曲线在3.83×104~3.83×109 copies/μL浓度范围内有良好的线性相关性(y= –3.287x+7.48, R2= 0.998)，大菱鲆弧菌在2.87×104~2.87×109 copies/μL浓度范围内有良好的线性相关性(y= –3.549x+8.14, R2= 0.998)，鳗弧菌在1.39×104~1.39×109 copies/μL浓度范围内有良好的线性相关性(y= –3.912x+7.83, R2= 0.999)，美人鱼发光杆菌美人鱼亚种在2.20×104~ 2.20×109 copies/μL浓度范围内有良好的线性相关性(y= –3.969x+7.07, R2=0.992)。

图 4 质粒标准品的扩增曲线和标准曲线 Fig.4 Amplification curve and standard curve of plasmid standard a：扩增曲线；b：标准曲线；1~6分别为109、108、107、106、105和104 copies /μL；Ⅰ：哈维氏弧菌；Ⅱ：副溶血弧菌；Ⅲ：大菱鲆弧菌；Ⅳ：鳗弧菌；Ⅴ：美人鱼发光杆菌美人鱼亚种 a: Amplification curve; b: Standard curve; 1~6 indicate 109, 108, 107, 106, 105 and 104 copies /μL, respectively; Ⅰ: V. harveyi; Ⅱ: V. parahaemolyticus; Ⅲ: V. scophthalmi; Ⅳ: V. anguillarum; Ⅴ: P. damselae subsp. damselae
2.4 集成芯片引物交叉反应分析

将5对引物集成到同一芯片上，进行引物间交叉反应分析。结果显示，5对引物在同一芯片上交叉反应影响较小，在25个反应循环数内不影响结果判读(图 5)。

图 5 引物交叉反应的微流控荧光定量PCR分析结果 Fig.5 Results of cross reaction analysis through microfluidic fluorescence quantitative PCR 荧光信号强证明发生阳性反应。Ⅰ：哈维氏弧菌；Ⅱ：鳗弧菌；Ⅲ：美人鱼发光杆菌美人鱼亚种；Ⅳ：副溶血弧菌；Ⅴ：大菱鲆弧菌；1、6：哈维氏弧菌引物；2、7：鳗弧菌引物；3、8：美人鱼发光杆菌美人鱼亚种引物；4、9：副溶血弧菌引物；5、10：大菱鲆弧菌引物；6~10：空白对照。 The apparent turbidity of the channel was positive reaction. Ⅰ: V. harveyi; Ⅱ: V. anguillarum; Ⅲ: P. damselae subsp. damselae; Ⅳ: V. parahaemolyticus; Ⅴ: V. scophthalmi; 1, 6: Primers of V. harveyi; 2, 7: Primers of V. anguillarum; 3, 8: Primers of P. damselae subsp. damselae; 4, 9: Primers of V. parahaemolyticus; 5, 10: Primers of V. scophthalmi; 6~10: Negative control.
2.5 多重微流控荧光定量PCR对5种病原菌的检测灵敏度

将5种病原菌的特异性引物集成到同一个微流控芯片上，分别用哈维氏弧菌、副溶血弧菌、大菱鲆弧菌、鳗弧菌和美人鱼发光杆菌美人鱼亚种DNA作为模板，在优化好的体系和反应条件下，同时进行微流控荧光定量PCR扩增，验证该方法的灵敏度。实验结果如图 6，集成后的微流控芯片对上述5种病原菌的最低检测限分别为40 CFU/mL (2.0×10–3 ng/μL)、20 CFU/mL (2.0×10–3 ng/μL)、200 CFU/mL (2.0× 10–2 ng/μL)、500 CFU/mL (1.0×10–2 ng/μL)、20 CFU/mL (1.0×10–3 ng/μL)。因此，判定集成后的芯片对5种病原菌的最低可检限为100 CFU/mL (0.01 ng/μL)，具有较高的灵敏度。

图 6 微流控荧光定量PCR集成芯片对5种病原菌同步检测的灵敏度验证结果 Fig.6 Sensitivity of multiplex microfluidic quantitative PCR on the 5 pathogenic bacteria Ⅰ~Ⅵ分别为105、104、103、102、101和100 CFU/mL的菌液；1：哈维氏弧菌；2：副溶血弧菌；3：美人鱼发光杆菌美人鱼亚种；4：鳗弧菌；5：大菱鲆弧菌；6~10：空白对照。 Ⅰ~Ⅵ indicated 105, 104, 103, 102, 101 and 100 CFU/mL bacteria suspension, respectively; 1: V. harveyi; 2: V. parahaemolyticus; 3: P. damselae subsp. damselae; 4: V. anguillarum; 5: V. scophthalmi; 6~10: Negative control.
2.6 多重微流控荧光定量PCR检测的临床应用效果验证

选取大菱鲆、凡纳滨对虾、仿刺参为实验对象，验证5种病原菌在大菱鲆(肌肉、鳃、肝)、凡纳滨对虾(肌肉、鳃)、仿刺参(呼吸树、肠道)组织中的检出效果，筛选出最佳检测部位。应用已建立的多重微流控荧光定量PCR检测方法对含有5种病原菌的上述组织进行样品检测，同时应用实验室常规实时荧光定量PCR进行同步检测作对比。结果如表 3所示，普通实时荧光定量PCR检测样品结果分别为100% (21/21)、100% (21/21)、100% (21/21)、100% (21/21)、95.2% (20/21)。所建立的多重微流控荧光定量PCR检测方法的准确率达到普通实时荧光定量PCR检测方法的96.2%以上，实验中未加菌液的组织检测结果均为阴性。样品平均检测时间仅为26 min，检测时长较普通实时荧光定量PCR反应时间(1 h 40 min)明显缩短。

表 3 微流控荧光定量PCR检测不同组织中5种病原菌的应用效果 Tab.3 Results of microfluidic fluorescence quantitative PCR in different tissues of 5 pathogens
3 讨论

近年来，病害频发导致了水产养殖业严重的经济损失，2020年我国水产病害损失约为589亿元，达到了历史最高值(崔利锋, 2021)。哈维氏弧菌、副溶血弧菌、大菱鲆弧菌、鳗弧菌和美人鱼发光杆菌美人鱼亚种是目前文献报道相对频繁的几种海水养殖主要致病菌，可感染鱼类(Qi et al, 2021; 兰欣等, 2020; 靳晓敏等, 2015; Mei et al, 2010; Kim et al, 2013)、凡纳滨对虾(Paria et al, 2021; 郑国兴等, 1990)和贝类(刘小莉等, 2017; Hidalgo et al, 2008)等多种海水经济动物。此外，美人鱼发光杆菌美人鱼亚种对鲸鱼和海豚等海洋哺乳动物(Rivas et al, 2013)以及人类也有致病性(Alvarez et al, 2006)。加强对上述病原菌的快速检测技术研究，可为防控这些病原菌的感染和传播提供重要的技术支撑。

目前，对病原菌的检测技术研究通常选用管家基因和毒力基因作为靶基因。本研究分别选取了哈维氏弧菌的vhhA基因、副溶血弧菌的toxR基因、大菱鲆弧菌的luxR基因、鳗弧菌的empA基因和美人鱼发光杆菌美人鱼亚种的Mcp基因作为目标基因开展快速检测技术研究。vhhA基因是哈维氏弧菌的溶血素编码基因，最早在高致病性毒株VIB 645中被发现(Zhang et al, 2005)，在NCBI上Blast比对发现该基因序列与哈维氏弧菌簇下其他菌种的溶血素基因相似度在87%以下。毒力基因toxR最先在霍乱弧菌(Vibrio cholerae)中被报道，后来陆续在副溶血弧菌、创伤弧菌(Vibrio vulnificus)、鳗弧菌、拟态弧菌(Vibrio mimicus)、溶藻弧菌(Vibrio alginolyticus)等多种弧菌中证实了该基因的存在(Lin et al, 1993)。有研究发现，异种细菌之间的toxR基因序列相似性较低，如副溶血弧菌和溶藻弧菌的toxR基因同源性仅为61.7%，可在种的水平上用于检测副溶血弧菌(Kim et al, 1999)。luxR基因是家族蛋白调控基因，广泛存在于革兰氏阴性菌中(Chaparian et al, 2016)。高志鑫等(2015)以大菱鲆弧菌的luxR基因为靶点设计LAMP扩增引物，建立了大菱鲆弧菌LAMP检测方法。鳗弧菌的empA是金属蛋白酶的编码基因，金属蛋白酶被认为是鳗弧菌侵入宿主体内不可缺少的重要因子(刘小莉等, 2017)。将病原菌特有的金属蛋白酶基因作为靶基因来检测病原菌，具有灵敏度高、特异性强等优势(余俊红等, 2002)。Mcp基因是美人鱼发光杆菌美人鱼亚种特有基因，可作为美人鱼发光杆菌美人鱼亚种检测的靶基因(Zhang et al, 2021)。本研究分别选用这5个基因作为检测目标菌的靶基因，均具有良好的种间特异性，为5种病原菌多重微流控荧光定量PCR检测方法的建立奠定了基础，保障所建立的检测技术具有较高的特异性和灵敏度。

微流控芯片最早由Manz等(1990)提出，是通过细微加工技术将微型功能元件组建成具有微流体控制的集成分析系统，具有自动化、集成化、便携化、成本低和快速化等特点(朱婧旸等, 2021)。本实验室在前期的研究中，已经针对单一的病原菌开展了微流控快速检测技术的研究(陈京等, 2020; Zhang et al, 2021)。而将不同病原菌的反应体系和反应条件在同一芯片进行集成化，可以满足多样品和多病原的实时、快速检测需求，显著提高检测效率，进而可开发应用于养殖现场的多病原微流控同步快速检测技术。余东阳等(2020)结合微流控与荧光定量PCR技术建立了寨卡病毒(ZIKV)、登革病毒(DENV)、黄热病毒(YFV)和基孔肯雅病毒(CHIKV)的快速核酸检测方法，25 min内即可完成1次上述4种病毒的同步快速检测，适用于现场检测与早期诊断。El-tholoth等(2021)基于微流控装置建立了环介导等温扩增(RT-LAMP)技术，实现了猪流行性腹泻病毒(PEDV)、传染性胃肠炎病毒(TGEV)和猪三角洲冠状病毒(PDCoV)的同步检测，耗时仅需30 min。但在水产领域，微流控检测技术研究相对较少，也未开展实践应用。本研究基于UF-150微流控荧光定量PCR仪平台，通过对微流控芯片的集成应用，建立了针对水产动物5种常见病原菌的多重微流控荧光定量PCR检测方法，实现了在同一检测芯片特异性扩增哈维氏弧菌、副溶血弧菌、大菱鲆弧菌、鳗弧菌和美人鱼发光杆菌美人鱼亚种5种病原菌，并且具有较高的灵敏度。通过应用所建立的多重微流控荧光定量PCR检测方法对水产动物样品进行临床检测，以及与实验室普通荧光定量PCR检测准确率进行对比，发现该方法检测动物组织样品的准确率可达到实验室普通实时荧光定量PCR检测方法准确率的96.2%以上，但检测时间只需26 min左右，较实验室普通实时荧光定量PCR的检测时间明显缩短，为进一步开发实用化的水产多病原现场快速检测技术奠定了工作基础。

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