海洋渔业2025，Vol.47Issue(4)：517-523,7.
大黄鱼寄生性盾纤毛虫PCR检测方法的建立与应用
Establishment and application of a PCR detection method for parasitic scuticociliates in Larimichthys crocea
摘要
Abstract
Scuticociliatosis,a disease caused by parasitic scuticociliates,has emerged as a critical threat to the fries of Larimichthys crocea,leading to substantial economic losses in aquaculture.To establish a rapid and precise etiological diagnosis method for the dection of scuticociliates,a polymerase chain reaction(PCR)-based detection method targeting the variable regions of the 18S rRNA gene in scuticociliates was developed.A pair of specific primers SSU244-R(5'-GTGAATCATAGTAACTGATCGA-3')and SSU1117-F(5'-TCCTTTAAGTTTCAGCCTTGCG-3')was designed and optimized to establish a robust PCR protocol.The 18S rRNA fragment of Miamiensis avidus was amplified using these primers and subsequently cloned into the pMD19-T vector to construct the positive standard.The optimized PCR conditions were as follows:initial denaturation at 95℃for 3 min,an additional 30 cycles(95℃for 30 s,54℃for 30 s,72℃for 1 min),and a final extension at 72℃for 7 min.Then,the sensitivity,specificity and diagnostic validity of the method were thoroughly validated.The primer pair exhibited high specificity towards scuticociliates,successfully amplifying DNA templates from Miamiensis avidus,Pseudoconchocelis globularis,Uronema marina,Metanophrys sinensis,Parauronema floridense,and Eminentia aciclata,while showing no cross-reactivity with non-target pathogens such as Cryptocaryon irritans,Amyloodinium ocellatum,Pseudomonas plecoglossicida,or DNA from healthy L.crocea.Sensitivity assays demonstrated a detection limit of 6.56×104 copies,confirming the method's capacity for identifying low-abundance infections.In practical applications,the PCR method detected scuticociliates in 5 out of 10 diseased fish samples,outperforming conventional microscopic examination,which identified only 4 positive cases.The enhanced sensitivity proved particularly advantageous for detecting early-stage or subclinical infections.This study successfully establishes a rapid,specific,and sensitive PCR detection method for scuticociliates,which can serve as a valuable tool for epidemiological investigations and early,rapid diagnosis of scuticociliatosis.关键词
大黄鱼/盾纤毛虫/PCR检测/核糖体小亚基基因Key words
Larimichthys crocea/scuticociliates/PCR test/18S rRNA分类
农业科技引用本文复制引用
池洪树,柯翎,林能锋,方勤美,陈永聪,施少华..大黄鱼寄生性盾纤毛虫PCR检测方法的建立与应用[J].海洋渔业,2025,47(4):517-523,7.基金项目
福建省公益类科研院所基本科研专项(2021R1027008) （2021R1027008）
福建省科技重大专项(2021NZ033025) （2021NZ033025）
宁德市揭榜挂帅攻关项目(ND2023J005) （ND2023J005）
福建省自然科学基金(2021J01487) （2021J01487）
