[目的]基于TaqMan-MGB探针建立虾肝肠胞虫(EHP)荧光定量PCR检测方法,为EHP的快速定量检测及实时监测提供技术手段.[方法]以EHP的SSU rDNA基因为检测靶基因,在其保守区设计特异性的扩增引物和TaqMan-MGB探针,将PCR扩增目标片段克隆至pMD18-T载体构建重组质粒pMD18-T-SSUEHP(标准品),以标准品DNA为模板优化反应体系,建立检测EHP的TaqMan-MGB探针荧光定量PCR;以10倍梯度稀释的标准品DNA为模板构建扩增标准曲线,并进行特异性、敏感性、重复性及临床应用试验.[结果]制备的重组质粒pMD18-T-SSUEHP DNA稳定性好,可满足作为标准品和阳性对照的要求;标准品(pMD18-T-SSUEHP)起始模板范围为2.0×101～2.0×109拷贝/反应时,建立的标准曲线线性关系良好,对应的线性回归方程为y=-3.232×lg(x) +39.51.基于TaqMan-MGB探针建立的EHP荧光定量PCR对标准品的检测灵敏度可达20拷贝/反应,对临床虾样品EHP的检测灵敏度约10拷贝/mg,较农业农村部推荐的套式PCR约提高10倍;对虾类其他常见病原均无扩增反应,其组内和组间变异系数分别为0.26％～0.72％和0.56％～0.92％,整个检测过程可在1h内完成.采用建立的TaqMan-MGB探针荧光定量PCR与套式PCR同时对276份临床虾样品进行检测比较,发现两种方法的符合率为96.7％,检测低拷贝数虾样品时前者具有更高的灵敏度;2018和2019年广西虾样品的EHP阳性检出率分别为16.2％和30.6％.[结论]基于TaqMan-MGB探针建立的EHP荧光定量PCR检测方法具有特异性强、灵敏度高、重复性好、定量范围宽及简单快速等优点,适用于虾类样品的EHP定量检测,可为EHP实时监测及EHPD快速诊断提供一种新的技术手段.

To investigate the effects of dietary carbohydrate on growth performance, body composition and digestive enzyme activities of juvenile crab, Scylla paramamosain, five isonitrogenous and isolipidic diets with varying carbohydrate contents of 20.34％, 25.59％, 30.17％, 35.30％ and 39.95％ were fed to juvenile crabs with an average body weight of (38.11±0.55) mg of triplicate groups, respectively, for a 3-week feeding trial. Results showed that the survival rate, weight gain ratio and specific growth rate of the juvenile crabs significantly increased firstly and then decreased under the increased dietary carbohydrate level (P<0.05) with the highest level at 30.17％. With the increasing dietary carbohydrate level, the body crude protein and body ash contents of the crabs were significantly increased firstly and then decreased (P<0.05) with the highest level at 35.30％. Dietary carbohydrate level significantly affected amylase activity (P<0.05) without any significant influence on protease and lipase activities (P>0.05). The amylase activity significantly induced by high carbohydrate level (P<0.05). These results indicate that the diet with 25.59％-35.30％ carbohydrate could greatly support growth performance. The regression model analysis showed that the most suitable dietary carbohydrate level reach the highest weight gain ratio is 29.93％.

介绍了对虾白斑综合征病毒(WSSV)的一般结构、危害及典型发病特征等研究进展;综述了消毒剂的使用(包含消毒剂的类型、优缺点、杀菌机理及其发展趋势等),中草药的分类及特点,并从药物作用和免疫效果2个方面阐述其在水产养殖业上的应用价值;益生菌的特点与优势,及其在水产养殖方面的应用;RNAi和疫苗在WSSV防治方面的研究进展以及健康养殖措施等白斑综合征病毒的防治技术.