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| 唇形后口虫(Boveria labialis) SYBR Green I 实时荧光定量 PCR 检测方法的建立及其应用 |
| Establishment of SYBR Green I real-time fluorescence quantitative PCR for parasitic Boveria labialis from the sea cucumber Apostichopus japonicus |
| 投稿时间：2024-03-22 修订日期：2024-04-06 |
| DOI： |
| 中文关键词: 刺参 寄生性疾病 唇形后口虫 实时荧光定量 PCR 检测 传播途径 |
| 英文关键词: Apostichopus japonicus Parasitic disease Boveria labialis Real-time fluorescent quantitative PCR detection Transmission route |
| 基金项目:山东省重点研发计划(重大科技创新工程)(2023CXGC010410)，中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金项目(2023TD29)和青岛市重点研发计划课题(22-3-3-hygg-1-hy)项目 |
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| 中文摘要: |
| 后口虫病是近年来影响刺参养殖效益的重要病害种类之一。唇形后口虫(Boveria labialis)是刺参后口虫病的病原。该物种快速、准确的检测方法的缺乏限制了对该病原的传播途径进行系统分析。本研究以测序获得的唇形后口虫部分线粒体基因组序列为基础，根据 nad10 基因序列设计唇形后口虫引物并建立其 SYBR Green I 实时荧光定量 PCR 检测方法，并对刺参不同养殖区和不同养殖模式下养殖系统唇形后口虫载量进行检测分析。结果表明，设计的唇形后口虫引物在质粒标准品 4.05×101~4.05×109 copies/μL 范围内建立的标准曲线具有良好的线性关系，相关系数 R2=0.997，熔解曲线呈现单一峰，无引物二聚体或非特异性扩增；灵敏度试验最低检测限为 40.5 copies/μL；所设计的引物仅对唇形后口虫出现特异性扩增，对海洋尾丝虫(Uronema marinum)、贪食纤口虫(Chaenea vorax)、僧帽肾形虫(Colpoda cucullus)和多小核草履虫(Paramecium multi-micronuleatum)无交叉反应；重复性试验中各浓度的批内和批间 Ct 值均一性较高，批内试验 CV 值为 0.32%~0.82%，批间试验 CV 值为 0.40%~0.88%，稳定性较好。利用本方法对四种刺参养殖模式不同养殖区的环境样品与饲料进行检测，结合镜检结果对比分析发现：海水中唇形后口虫 DNA 载量与刺参体内唇形后口虫量呈中度正相关(r=0.563)，底泥、附着物样品中的唇形后口虫 DNA 载量与刺参体内唇形后口虫量呈高度正相关(r=0.931)，推测鲜海泥是唇后口虫传播的重要载体之一。相关研究结果为唇形后口虫的快速检测、传播途径解析和防控提供参考。 |
| 英文摘要: |
| Boveria disease is one of the important diseases affecting the aquaculture efficiency of sea cucumber in recent years. Boveria labialis is the pathogen of Boveria disease of Apostichopus japonicus. Due to the lack of rapid and accurate detection methods, it is difficult to analyze the transmission route of the pathogen. In this study, based on the partial mitochondrial genome sequence of B. labialis obtained by sequencing, primers were designed according to the nad10 gene sequence, and SYBR Green I real-time fluorescent quantitative PCR detection method was established. The concentration of B. labialis in different culture areas and different culture modes of A. japonicus was detected and analyzed. The results showed that the standard curve established by the designed primers had a good linear relationship in the range of plasmid standard 4.05×101~4.05×109 copies/μL, with the reliability (R2) was 0.997. The melting curve showed a single peak, without primer dimer or nonspecific amplification. The minimum detection limit of sensitivity test is 40.5 copies/μL. The designed primers only showed specific amplification for B. labialis, but had no cross reaction for Uronema sp., chaenea sp., colpoda sp. and Paramecium sp. In the repeatability test, the homogeneity of Ct values in the intra-assay and inter-assay of each concentration was high, the CV values of intra-assay and inter-assay tests were 0.32%~0.82% and 0.40%~0.88%, respectively, showing good stability. This method was used to detect the environmental samples and feeds in different culture areas of four kinds of A. japonicus culture modes. Combined with the comparative analysis of microscopic examination results, it was found that there was a moderate positive correlation (r=0.563) between the DNA load of B. labialis in sea water and the infection degree of B. labialis in A. japonicus, and there was a high positive correlation (r=0.931) between the DNA load of B. labialis in sediment and attachment samples and the infection degree of B. labialis in A. japonicus. It was speculated that fresh sea mud was one of the important carriers for the transmission of B. labialis. The relevant research results provide reference for the rapid detection, transmission route analysis and prevention and control of B. labialis. |
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