大口黑鲈锥体虫TaqMan探针荧光定量PCR检测方法的建立  

Establishment of a real-time qPCR detection method for Trypanosoma Micropterus

作　　者：徐锐龙 孙龑鑫 何晓茵 吴文豪 苏友禄 江飚 XU Ruilong;SUN Yanxin;HE Xiaoyin;WU Wenhao;SU Youlu;JIANG Biao(College of Marine Sciences,South China Agricultural University,Guangzhou 510642,China;College of Animal Science and Technology,Zhongkai University of Agriculture and Engineering,Guangzhou 510225,China;Innovative Institute of Animal Healthy Breeding,Zhongkai University of Agriculture and Engineering,Guangzhou 510225,China)

机构地区：[1]华南农业大学海洋学院,广东广州510642 [2]仲恺农业工程学院动物科技学院,广东广州510225 [3]仲恺农业工程学院健康养殖创新研究院,广东广州510225

出　　处：《仲恺农业工程学院学报》2025年第2期6-11,共6页Journal of Zhongkai University of Agriculture and Engineering

基　　金：国家自然科学基金项目(32102851)。

摘　　要：锥体虫病在珠三角大口黑鲈(Micropterus salmoides)养殖中频发,造成巨大经济损失.为开展大口黑鲈锥体虫(Trypanosoma micropterus)早期诊断,该研究以大口黑鲈锥体虫18S rDNA保守序列作为靶基因设计引物和TaqMan探针,建立了一种荧光定量检测方法,并对该方法的灵敏性、重复性和特异性进行评估.结果表明,该方法的循环阈值(Ct值)与锥体虫靶基因的质粒浓度(1.27×10^(1)~1.27×10^(8) copies/μL)呈显著的线性回归函数关系,R2为0.999 1,其扩增效率为99.9%.该方法最低可检测13 copies靶基因,具有稳定的重复性和良好的特异性,组内和组间变异系数均小于2%,对池塘多种生物和细菌均无扩增反应.通过检测发病大口黑鲈倍比稀释后的血液,可检测到1.4个锥体虫,其Ct值为31.38.综上,该方法具有良好的特异性、重复性和灵敏性,可为该病早期诊断和流行病学调查提供了技术支撑.Trypanosomiasis is one of the most important disease of Micropterus salmoides in the Pearl River Delta,which causes huge economic losses.To accurately predict disease outbreaks.A pair of specific primers and a TaqMan probe based on the Trypanosoma micropterus 18S rRNA gene were developed and used in a real-time PCR(qPCR).The sensitivity,repeatability and specificity of the method were evaluated.The cyclic threshold(Ct value)of the method demonstrated a significant linear regression relationship with the plasmid concentration of the target genes of T.micropterus(1.27×10^(1)~1.27×10^(8) copies/μL).The coefficient of determination(R^(2))value is 0.9991,and the amplification efficiency is 99.9%.The qPCR assay attained a limit of detection of 13 copies of the target gene,with stable repeatability and good specificity.The coefficient of variation within and between groups is less than 2%,and there is no amplification reaction to other biosome and bacteria from the ponds.Furthermore,the assay was used to detect a series of dilutions of diseased M.salmoides serum,and a limit of detection was 1.4 trypanosome with credible Ct value(31.38).This study provides a valuable tool for assisting in the early monitoring and epidemiological investigation in aquaculture.

关 键 词：大口黑鲈(Micropterus salmoides) 锥体虫(Trypanosoma) 18S rDNA基因 TAQMAN探针 荧光定量PCR 

分 类 号：S942.92[农业科学—水产养殖]