| 郝文悦,王锦锦,葛建龙,李彬,王印庚,廖梅杰,荣小军,赵宏晶,江敏棋,赵文广,牛立成,潘娇.唇形后口虫SYBR GreenⅠ实时荧光定量PCR检测方法的建立及其应用.渔业科学进展,2025,46(3):183-193 |
| 唇形后口虫SYBR GreenⅠ实时荧光定量PCR检测方法的建立及其应用 |
| SYBR GreenⅠreal-time fluorescence quantitative PCR for parasitic Boveria labialis identification from the sea cucumber Apostichopus japonicus |
| 投稿时间：2024-03-22 修订日期：2024-04-06 |
| DOI：10.19663/j.issn2095-9869.20240322002 |
| 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(2023TD29)和青岛市重点研发计划(22-3-3-hygg-1-hy)共同资助 |
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| 中文摘要: |
| 后口虫病是近年来影响刺参(Apostichopus japonicus)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。由于缺乏针对该物种的快速、准确的检测方法，研究人员难以系统解析该病原体的传播途径。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，建立其SYBR GreenⅠ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中唇形后口虫载量进行检测分析。结果显示，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；灵敏度实验最低检测限为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；重复性实验中各浓度的批内和批间Ct值均一性较高，批内实验变异系数(CV)值为0.32%~0.82%，批间实验CV值为0.40%~0.88%，稳定性较好。利用本方法对4种刺参养殖模式不同养殖区的环境样品和饲料进行检测，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，鲜海泥是唇形后口虫传播的重要载体之一。本研究结果可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 affects the aquaculture efficiency of sea cucumbers. Boveria labialis causes Boveria disease in Apostichopus japonicus. Owing to the lack of rapid and accurate detection methods, analyzing the transmission route of the pathogen remains complicated. Based on the partial mitochondrial genome sequence of B. labialis obtained using sequencing, primers were designed to amplify nad10 and a SYBR GreenⅠreal-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 standard curve established by the designed primers had a good linear relationship in the range of the plasmid standard of 4.05×101–4.05×109 copies/µL, with a reliability (R2) of 0.997. The melting curve showed a single peak, without primer dimers or nonspecific amplification. The minimum detection limit of sensitivity test was 40.5 copies/µL. The designed primers showed specific amplification for B. labialis only and exhibited no cross reaction to 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- and inter-assay tests were 0.32%–0.82% and 0.40%–0.88%, respectively, indicating good stability. This method was used to detect 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, 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. A high positive correlation (R=0.931) was found between the DNA load of B. labialis in sediment and attachment samples and the infection degree of B. labialis in A. japonicus. Fresh sea mud was an important carrier for B. labialis transmission. The relevant research results provide reference for the rapid detection, transmission route analysis, and B. labialis prevention and control. |
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