摘要
为建立同时检测淡水鱼中华支睾吸虫、东方次睾吸虫和日本全冠吸虫的三重PCR检测方法,本研究根据GenBank中登录的各吸虫参考序列中保守区域设计3对特异性引物,通过优化反应条件,建立了3种吸虫囊蚴的多重PCR检测方法,并利用该方法对52尾淡水鱼样品中吸虫囊蚴的感染情况进行检测。结果显示,本研究所建立的方法对东方次睾吸虫、华支睾吸虫、日本棘隙吸虫能分别扩增到508 bp、311 bp和208 bp的目的条带,对鸭对体吸虫、舟形嗜气管吸虫、横川后殖吸虫和卷棘口吸虫的基因扩增结果均为阴性,特异性较强;敏感性试验结果显示,该方法对3种吸虫囊蚴重组质粒标准品检测下限均为104拷贝/μL。利用该三重PCR方法对淡水鱼临床样品进行检测,结果显示该方法与形态学检测方法结果一致。本研究在国内首次建立了淡水鱼3种吸虫囊蚴多重PCR检测方法,为鱼源性吸虫病的快速检测及鉴别诊断提供了技术手段,为鱼源性寄生虫病的防控奠定了基础。
To establish a multiplex-PCR method for detection of three kinds of Trematode metacercariae in freshwater fish,three pairs of specific primers were designed based on the conservative region of reference sequences deposited in GenBank.By optimizing the reaction conditions,a multiplex PCR method was established,and 52 freshwater fish samples were checked by this method.The results showed that the multiplex PCR method established in this study was highly specific.PCR products at 508 bp,311 bp and 208 bp could be amplify from Metorchis orientalis,Clonorchis sinensis,and Holostephanus dubinini,respectively,while Amphimerus anatis,Tracheophilus cymbius,Metagonimus yokogawai and Echinostoma revolutum could not be detected.Sensitivity test showed that the multiplex PCR method can detected the minimum concentration at 104 copies/μL.The multiplex PCR was used to detect 52 freshwater fish samples,and the results were consistent with those of morphology identification.In this study,the multiplex PCR method could detect three kinds of trematode metacercariae in freshwater fish with high sensitivity and specificity,thus the method may provide technical support for rapid detection and differential diagnosis,which has important significance for the prevention and control of fish-borne trematode diseases.
作者
高俊峰
王鑫
毛瑞锋
孙云异
王春仁
黄翠琴
GAO Jun-feng;WANG Xin;MAO Rui-feng;SUN Yun-yi;WANG Chun-ren;HUANG Cui-qin(College of Life Science,Longyan University/Fujian Provincial Key Laboratory for the Prevention and Control of,Animal Infectious Diseases and Biotechnology,Longyan 364012,China;College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2022年第2期157-161,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(32172886)
黑龙江省自然科学基金项目(LH2021C071)
福建省家畜传染病防治与生物技术重点实验室开放基金项目(2019KF04)
黑龙江省博士后面上资助(LBH-Z19191)。
