摘要
【背景】2023年至2025年,锥体虫(Trypanosoma spp.)严重威胁着大黄鱼(Lar-imichthys crocea)产业的健康发展,造成巨大的经济损失。目前,对于大黄鱼锥体虫病的防控措施有限,早期诊断对防控至关重要,但现有检测方法存在灵敏度低、操作繁琐等局限。【目的】建立大黄鱼锥体虫快速精准检测技术,探究其组织分布特征,为病害早期诊断和流行病学调查提供技术支持。【方法】基于大黄鱼源锥体虫18S rDNA保守序列设计特异性引物与TaqMan探针,构建荧光定量PCR(qPCR)方法;通过重组质粒梯度稀释建立标准曲线,验证方法的特异性、灵敏度和重复性;采集三都岛、霞浦、连江养殖区大黄鱼样品,检测感染率并分析9种组织中锥体虫含量差异。【结果】该方法标准曲线线性关系良好(R2=0.997),检测下限达25拷贝/µL;组间与组内循环阈值变异系数分别<2.0%和<1.5%,重复性稳定;对车轮虫(Trichodina)、副溶血弧菌(Vibrio parahaemolyticus)、神经坏死病毒(NNV)等常见水产病原无交叉反应,特异性显著。60尾样品总体感染率为93.33%,三都岛感染率(100%)最高;组织分布显示,血液、心脏、脾脏中锥体虫含量极显著高于鳃、肝脏、肾脏等其他组织(P<0.01);此外,鲻鱼(Mugilcephalus)、真鲷(Pagrusmajor)中仅少量感染且病原含量极低;非鱼类样品中裙带菜(Undariapinnatifida)检出率(93.33%)最高,网衣、福建牡蛎(Crassostrea angulata)检出少量,其余未检出。【结论】本研究建立的荧光定量PCR方法具有高特异性、灵敏性和重复性,可用于大黄鱼锥体虫病的早期诊断;锥体虫在血液、心脏、脾脏中富集的组织趋向性,为解析其致病机制、制定针对性防控策略提供了科学依据,对保障大黄鱼养殖产业健康发展具有重要意义。
[Introduction]From 2023 to 2025,Trypanosoma spp.have emerged as a significant threat to the sustainable aquaculture of Larimichthys crocea,causing substantial economic losses.Current strategies for con-trolling andpreventing trypanosomiasis in thisspecies remain limited.Early diagnosis is crucial for disease pre-vention and control,but existing detection methods have limitations such as low sensitivity and cumbersome op-eration.[Objective]This study aims to establish a rapid and accurate detection technology for Trypanosoma spp.in L.crocea and explore its tissue distribution characteristics,providing support for early disease diagnosis and epidemiological investigation.[Methods]Specific primers and TaqMan probes were designed based on the conserved sequence of Trypanosoma spp.18S rDNA in GenBank,and a fluorescence quantitative PCR(qPCR)method was constructed;a standard curve was established through gradient dilution of recombinant plasmids to verify the specificity,sensitivity,and repeatability of the method;samples of L.crocea from Sandu Island,Xiapu,and Lianjiang breeding areas were collected to detect infection rates and analyze the differences in Tryp-anosoma spp.content in 9 tissues.[Results]The standard curve showed a good linear relationship(R2=0.997),with a detection limit of 25 copies/µL;the inter-group and intra-group cycle threshold variation coefficients were respectively<2.0%and<1.5%,and the repeatability was stable;there was no cross-reaction with common aquatic pathogens such as ciliates,Vibrio parahaemolyticus,and neurotropic virus,and the specificity was signi-ficant.The overall infection rate of 60 samples was 93.33%,with the highest infection rate in Sandu Island(100%);the tissue distribution showed that the content of Trypanosoma spp.in blood,heart,and spleen was sig-nificantly higher than that in gills,liver,and kidneys,etc.(P<0.01).In addition,only a small number of Mugil cephalus and Pagrus major were infected,with extremely low levels of pathogens.Among non-fish samples,Undaria pinnatifida(Wakame)had the highest detection rate(93.33%),while a small amount of pathogens were detected in netting and oysters,and no pathogens were detected in the remaining samples.[Conclusion]The fluorescence quantitative PCR method established in the study has high specificity,sensitivity,and repeatability,and can be used for early diagnosis of Trypanosoma spp.disease;the tendency of Trypanosoma spp.enrich-ment in tissues such as blood,heart,and spleen provides a scientific basis for analyzing its pathogenic mechan-ism and formulating targeted control strategies,and is of great significance for the healthy development of the L.crocea aquaculture industry.
作者
葛辉
郭香
巫旗生
宁岳
叶军
吴丽云
谢鑫怡
张东玲
黄强
王为刚
GE Hui;GUO Xiang;WU Qisheng;NING Yue;YE Jun;WU Liyun;XIE Xinyi;ZHANG Dongling;HUANG Qiang;WANG Weigang(Key Laboratory of Cultivation and High-Value Utilization of Marine Organisms in Fujian Province,Fisheries Research Institute of Fujian,Xiamen 361013,China;Fisheries College,Jimei University,Xiamen 361021,China;Xiapu Center for Marine and Fisheries Development,Ningde 355100,China;Fisheries Technology Extension Center of Lianjiang County,Fuzhou 350599,China)
出处
《渔业研究》
2025年第5期651-660,共10页
Journal of Fisheries Research
基金
福建省促进海洋与渔业产业高质量发展专项(FJHYF-L-2025-02-012)
大黄鱼种业创新与产业化开发(2021FJSC2Y01)
福建省海洋与渔业综合服务专项(FYZF-KTYJ-2025-10、FYZF-BHZL-2025-2)。
关键词
大黄鱼
锥体虫
荧光定量PCR
检测方法
组织分布
Larimichthys crocea
Trypanosoma spp.
fluorescence quantitative PCR
detection method
tissue distribution
作者简介
第一作者:葛辉,男,副研究员,研究方向为海洋经济动物疾病防控。E-mail: gehuizlj@163.com。
