水产养殖水体无乳链球菌实时荧光定量PCR检测方法的建立

IF 0.7 4区 农林科学 Q4 FISHERIES
Zaiyong Si, Zhenhua Ma, Fuguang Luo, Jingu Shi, Jie Huang, Zhiqiang Wang, Yanhong Wen, Yi Yi, Shuyu Han
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引用次数: 0

摘要

无乳链球菌对罗非鱼养殖有严重的负面影响,快速检测养殖水体中微量无乳链球菌并采取及时有效的灭菌措施可显著降低其在罗非鱼养殖中爆发的概率。本研究基于无乳链球菌CAMP因子(cfb) (GU217532.1)序列,建立了罗非鱼养殖水体中无乳链球菌的荧光定量检测方法。结果表明,Ct值与质粒拷贝数的对数呈良好的线性关系(y =−3.49x + 38.78;R2 = 0.997),质粒浓度范围为1.57 × 102 ~ 1.57 × 109 copies/μL,建立的方法灵敏检测的最低浓度为1.57 × 102 copies/μL。该方法检测无乳链球菌的特异性高,以维罗氏气单胞菌、表皮葡萄球菌、嗜水气单胞菌和迟发爱德华菌的DNA为模板进行qPCR时均为阴性。组内和组间重复实验变异系数均小于2%,表明该方法具有较高的稳定性和特异性。应用该方法对两种罗非鱼养殖水体中的无乳链球菌进行了检测。结果表明,两种水样中无乳链球菌的浓度分别为12拷贝/mL和2拷贝/mL。该方法可直接检测出养殖水体中痕量无乳链球菌,便于罗非鱼养殖中无乳链球菌的诊断、防治,将大大降低无乳链球菌大规模爆发的概率和经济损失。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Establishment of SYBR Green I Real-Time PCR for Detection of Streptococcus agalactiae in Aquaculture Waters
Streptococcus agalactiae has a serious negative impact on tilapia aquaculture, and rapid detection of trace S. agalactiae in aquaculture waters and timely and effective sterilization measures could significantly reduce the probability of its outbreak in tilapia farming. Here, we established a fluorescence quantitative detection method of S. agalactiae in tilapia aquaculture waters based on the Streptococcus agalactiae CAMP factor (cfb) (GU217532.1) sequence of its CAMP factor. The results showed that the Ct value and logarithm of plasmid copy number presented a good linear relationship (y = −3.49x + 38.78; R2 = 0.997) in the plasmid concentration range of 1.57 × 102−1.57 × 109 copies/μL, and the lowest concentration for sensitive detection of the established method was 1.57 × 102 copies/μL. The method exhibited high specificity for the detection of S. agalactiae and generated negative results when using the DNA of Aeromonas veronii, Staphylococcus epidermidis, Aeromonas hydrophila, and Edwardsiella tarda as templates for qPCR. Intra- and inter-group repeated experiments produced variation coefficients lower than 2%, indicating high stability and specificity of the method. The method was then used to detect S. agalactiae in two tilapia aquaculture waters. The results showed that the concentration of S. agalactiae was 12 copies/mL and 2 copies/mL in the two water samples, respectively. The method can directly detect trace S. agalactiae in the aquaculture water and facilitate the diagnosis, prevention, and control of S. agalactiae in tilapia aquaculture, which will greatly reduce the probability of large-scale outbreak of S. agalactiae and economic loss.
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来源期刊
Journal of Applied Ichthyology
Journal of Applied Ichthyology 生物-海洋与淡水生物学
CiteScore
2.30
自引率
11.10%
发文量
73
审稿时长
3-8 weeks
期刊介绍: The Journal of Applied Ichthyology publishes articles of international repute on ichthyology, aquaculture, and marine fisheries; ichthyopathology and ichthyoimmunology; environmental toxicology using fishes as test organisms; basic research on fishery management; and aspects of integrated coastal zone management in relation to fisheries and aquaculture. Emphasis is placed on the application of scientific research findings, while special consideration is given to ichthyological problems occurring in developing countries. Article formats include original articles, review articles, short communications and technical reports.
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