多子小瓜虫PCR和SYBR Green实时荧光定量PCR检测方法的建立及应用  

Establishment and application of PCR and SYBR Green real-time fluorescence quantitative PCR assays for detection of Ichthyophthirius multifiliis

作　　者：曹泽艺 周庆杰 陈凯[2] 习丙文[1,2] 谢骏[1,2] 潘良坤[2] 毛颖 CAO Zeyi;ZHOU Qingjie;CHEN Kai;XI Bingwen;XIE Jun;PAN Liangkun;MAO Ying(Wuxi Fisheries College,Nanjing Agricultural University,Wuxi 214081,China;Key Laboratory of Integrated Rice-Fish Farming Ecology,Ministry of Agriculture and Rural Affairs,Freshwater Fisheries Research Center,Chinese Academy of Fishery Sciences,Wuxi 214081,China;Aquatic and Animal Husbandry Station of Yixing City,Yixing 214252,China)

机构地区：[1]南京农业大学无锡渔业学院,江苏无锡214081 [2]中国水产科学研究院淡水渔业研究中心,农业农村部稻渔综合种养生态重点实验室,江苏无锡214081 [3]宜兴市水产畜牧站,江苏宜兴214252

出　　处：《水产学报》2024年第10期161-168,共8页Journal of Fisheries of China

基　　金：国家现代农业产业技术体系专项(CARS-45)。

摘　　要：为克服现有小瓜虫病的显微镜检和PCR诊断等方法在该寄生虫的感染早期阶段和环境样本检测中存在的缺陷,建立特异性强、灵敏度高的多子小瓜虫PCR检测方法,实验根据多子小瓜虫线粒体COⅠ序列设计和筛选了一对引物,经过PCR程序优化、特异性、灵敏性验证以及临床和环境样品检测分析,分别建立了普通PCR和荧光定量PCR检测方法。结果显示,本研究获得的检测引物对多子小瓜虫有较高的扩增特异性,对同属纤毛虫类的草履虫、四膜虫和肠袋虫以及常见养殖鱼类宿主异育银鲫、草鱼、尼罗罗非鱼和团头鲂等样本均无扩增;扩增特异性和灵敏度都优于文献报道的方法。其中,普通PCR最低检测的样本浓度为掠食体2.67个/μL,荧光定量PCR在掠食体0.02个/μL时依然能有效检出,荧光定量PCR检测灵敏度高于普通PCR。研究表明,在临床样本和养殖水环境样本检测应用中,基于该引物的两种PCR方法表现出很高的一致性,能有效检出潜伏感染鱼体和养殖水环境中的多子小瓜虫。本研究所建立的多子小瓜虫的检测方法特异性强、灵敏度高,适用于淡水养殖中小瓜虫病的早期诊断和病原体的检测。Ichthyophthirius multifiliis is the causative agent of“white spot”disease,which affects numerous freshwater fish and causes severe economic losses to worldwide aquaculture.The disease of I.multifiliis infection could develop very fast,and there are few practical measures to treat this heavy infection.To overcome the shortcomings of existing methods,for detecting I.multifiliis in the early stage and environmental samples,such as low sensitivity of visual diagnosis with microscope and low specificity of PCR methods developed in the previous studies,here we developed and validated novel PCR assays.In this study,a pair of primers(qIchF 5′-TTCTGCCCGTACTTTAGTTACC-3′and qIchR 5′-TGGTTGTACTAACACCTGCAA-3′)were designed and screened to target the mitochondrial COⅠgene of I.multifiliis.The length of the amplified product is 131 bp.After PCR programme optimization,specificity and sensitivity verification,clinical and environmental samples detection and analysis,standard PCR and real-time PCR assays were established,respectively.The standard PCR was incubated at 95℃for 3 min;35 cycles of denaturation at 95℃for 15 s,annealing at 51℃for 15 s,and extension at 72℃for 10 s;and final extension at 72℃for 5 min.For real-time PCR was incubated at 95℃for 30 s;40 cycles of denaturation at 95℃for 10 s,annealing and extension at 60℃for 30 s.The results showed that the primers obtained in this study had high amplification specificity for I.multifiliis.Ciliates Paramecium sp.,Tetrahymena sp.,and Balantidium sp.were not amplified,nor were common farmed fish hosts Carassius auratus gibelio,grass carp(Ctenopharyngodon idella),Nile tilapia(Oreochromis niloticus),and Megalobrama amblycephala.Amplification specificity and sensitivity were better than existing methods.The limit of detection for standard PCR was 2.67 theronts/μL,while real-time PCR could be detected at 0.02 theront/μL.Among them,the detection sensitivity of real-time PCR was higher than that of standard PCR.In the detection application of clinical sa

关 键 词：多子小瓜虫 PCR 荧光定量PCR 小瓜虫病 检测方法 

分 类 号：S941.5[农业科学—水产养殖]