Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; Shanghai Ocean University, Shanghai 201306, China.
Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China.
J Invertebr Pathol. 2018 May;154:95-101. doi: 10.1016/j.jip.2018.04.005. Epub 2018 Apr 13.
Shrimp hemocyte iridescent virus (SHIV) is a recently reported shrimp virus, which threats the cultured white-leg shrimp Litopenaeus vannamei and can cause huge economic loss in shrimp farming industry in China. A quantitative real time polymerase chain reactio (qPCR) assay was developed using a TaqMan probe to detect and quantify SHIV. A pair of qPCR primers, which amplify a 188 bp DNA fragment, and a TaqMan probe were selected from ATPase gene (ORF114R) of the SHIV genome. The primers and TaqMan probe used in this assay were shown to be specific for SHIV and did not react with White spot syndrome virus (WSSV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Hepatopancreatic parvovirus (HPV), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VP), and Enterocytozoon hepatopenaei (EHP), or healthy shrimp DNA. The detection limit of the qPCR method was as low as 4 copies per reaction. The diagnostic sensitivity and the diagnostic specificity of the qPCR compared with nested PCR were 95.3% and 99.2%, respectively. The resulting standard curves showed high correlation coefficient values (R = 0.998) in the range of 4 × 10 to 4 × 10 DNA copies/reaction. Test of farm samples showed that SHIV was detected in L. vannamei, Fenneropenaeus chinensis and Macrobrachium rosenbergii contained SHIV ranged from 1.21E+02 to 7.95E+07 copies (μg DNA). Quantitative detection of different tissues in artificial infected shrimp showed that haemolymph contained the highest SHIV load and muscle contained lowest SHIV load.
虾虹彩病毒 (SHIV) 是一种最近报道的虾病毒，它威胁着养殖的凡纳滨对虾 (Litopenaeus vannamei)，并可能给中国的对虾养殖业造成巨大的经济损失。本研究建立了一种基于 TaqMan 探针的实时荧光定量 PCR (qPCR) 方法来检测和定量 SHIV。根据 SHIV 基因组的 ATPase 基因 (ORF114R) 设计了一对 qPCR 引物和 TaqMan 探针，扩增片段长度为 188 bp。该检测方法具有高度特异性，与白斑综合征病毒 (WSSV)、传染性皮下及造血组织坏死病毒 (IHHNV)、对虾肝胰腺细小病毒 (HPV)、副溶血弧菌引起的急性肝胰腺坏死病 (VP)、对虾血细胞虹彩病毒 (EHP) 或健康虾的 DNA 均无交叉反应。该 qPCR 方法的检测限低至 4 个拷贝/反应。与巢式 PCR 相比，qPCR 的诊断灵敏度和诊断特异性分别为 95.3%和 99.2%。标准曲线的相关系数 (R²) 值在 4×10⁻⁴ 到 4×10⁻⁷ 拷贝/反应之间均高达 0.998。对养殖样品的检测结果表明，凡纳滨对虾、中国明对虾和罗氏沼虾中均检测到 SHIV，其拷贝数 (μg DNA) 范围为 1.21E+02 到 7.95E+07。对人工感染虾不同组织的定量检测结果显示，血淋巴中 SHIV 载量最高，肌肉中载量最低。
