本研究建立了一种对虾传染性皮下及造血组织坏死病毒(Infectious Hypodermal and Hematopoietic Necrosis Virus, IHHNV) TaqMan MGB探针荧光定量PCR检测方法。根据GenBank中已登录的IHHNV基因序列(GenBank登录号:AF218226)设计引物和...本研究建立了一种对虾传染性皮下及造血组织坏死病毒(Infectious Hypodermal and Hematopoietic Necrosis Virus, IHHNV) TaqMan MGB探针荧光定量PCR检测方法。根据GenBank中已登录的IHHNV基因序列(GenBank登录号:AF218226)设计引物和探针建立的检测方法,采用构建的pMD-18-IHHNV质粒建立的标准曲线为y = −0.3084X + 12.631,R2 = 0.9995。另外该定量检测方法的最低检出限为5 copies/μl,与对虾其它常见疫病均无交叉反应,且不同浓度标准品组内的变异系数(CV)在0.08%~0.26%之间,与普通PCR检测结果比较显示对虾样品的IHHNV阳性检出率提高,说明该方法具有良好的灵敏度、特异性和重复性,能够为对虾传染性皮下及造血组织坏死病的早期快速诊断提供技术支撑。This study established a contagious subcutaneous and hematopoietic tissue necrosis virus TaqMan in shrimp MGB probe fluorescence quantitative PCR detection method. The primers and probes for this method were designed based on the IHHNV gene sequence already registered in GenBank (GenBank accession number: AF218226). The standard curve was established using the constructed pMD-18-IHHNV plasmid, with the equation y = −0.3084X + 12.631 and R2 = 0.9995. In addition, the minimum detection limit of this quantitative detection method is 5 copies/μl. This method has no cross reactivity with other common diseases of shrimp. The coefficient of variation (CV) within different concentration standard groups of this method ranges from 0.08% to 0.26%. Compared with ordinary PCR detection results, the IHHNV positive detection rate of shrimp samples has increased. This method demonstrates good sensitivity, specificity, and reproducibility. This method can provide technical support for the early and rapid diagnosis of infectious subcutaneous and hematopoietic tissue necrosis disease in shrimp.展开更多
传染性皮下和造血器官坏死病毒(infectious hypodermal and hematopoietic necrosis virus,IHHNV)是对虾养殖的重要病原之一,可感染多种虾种。罗氏沼虾(Macrobrachium rosenbergii)是中国养殖的重要沼虾品种之一。根据国际兽疫局(Office...传染性皮下和造血器官坏死病毒(infectious hypodermal and hematopoietic necrosis virus,IHHNV)是对虾养殖的重要病原之一,可感染多种虾种。罗氏沼虾(Macrobrachium rosenbergii)是中国养殖的重要沼虾品种之一。根据国际兽疫局(Office International des Epizooties,OIE)推荐的IHHNV检测方法,在中国大陆地区首次开展IHHNV在罗氏沼虾中的感染和流行情况调查。结果显示,在研究区的罗氏沼虾养殖区,IHHNV广泛流行,阳性率高达90%;但所有成年罗氏沼虾均未表现出明显的病症,仅表现为病毒的携带。通过基因序列分析显示,检测到的华南地区毒株属于Ⅰ型感染株,与菲律宾株进化关系较为接近;华东地区毒株属于Ⅱ型感染株,与东南亚株进化关系较近。本研究为IHHNV在罗氏沼虾内的感染、流行和防控提供了详细参考。展开更多
A reverse transcription polymerase chain reaction (RT PCR) was used for detection of infectious hypodermal and haematopoietic necrosis virus (IHHNV) from the gills and subcutaneous tissue of Penaeus vannamei (10-15cm ...A reverse transcription polymerase chain reaction (RT PCR) was used for detection of infectious hypodermal and haematopoietic necrosis virus (IHHNV) from the gills and subcutaneous tissue of Penaeus vannamei (10-15cm in body size) without clinical signs. With primers 77012R and 77353F, a fragment of 356bp of IHHNV was amplified in RT PCR. The amplified product was sequenced and showed more than 98% homologue with the sequences of other IHHNV strains in Gene Bank.展开更多
文摘本研究建立了一种对虾传染性皮下及造血组织坏死病毒(Infectious Hypodermal and Hematopoietic Necrosis Virus, IHHNV) TaqMan MGB探针荧光定量PCR检测方法。根据GenBank中已登录的IHHNV基因序列(GenBank登录号:AF218226)设计引物和探针建立的检测方法,采用构建的pMD-18-IHHNV质粒建立的标准曲线为y = −0.3084X + 12.631,R2 = 0.9995。另外该定量检测方法的最低检出限为5 copies/μl,与对虾其它常见疫病均无交叉反应,且不同浓度标准品组内的变异系数(CV)在0.08%~0.26%之间,与普通PCR检测结果比较显示对虾样品的IHHNV阳性检出率提高,说明该方法具有良好的灵敏度、特异性和重复性,能够为对虾传染性皮下及造血组织坏死病的早期快速诊断提供技术支撑。This study established a contagious subcutaneous and hematopoietic tissue necrosis virus TaqMan in shrimp MGB probe fluorescence quantitative PCR detection method. The primers and probes for this method were designed based on the IHHNV gene sequence already registered in GenBank (GenBank accession number: AF218226). The standard curve was established using the constructed pMD-18-IHHNV plasmid, with the equation y = −0.3084X + 12.631 and R2 = 0.9995. In addition, the minimum detection limit of this quantitative detection method is 5 copies/μl. This method has no cross reactivity with other common diseases of shrimp. The coefficient of variation (CV) within different concentration standard groups of this method ranges from 0.08% to 0.26%. Compared with ordinary PCR detection results, the IHHNV positive detection rate of shrimp samples has increased. This method demonstrates good sensitivity, specificity, and reproducibility. This method can provide technical support for the early and rapid diagnosis of infectious subcutaneous and hematopoietic tissue necrosis disease in shrimp.
文摘传染性皮下和造血器官坏死病毒(infectious hypodermal and hematopoietic necrosis virus,IHHNV)是对虾养殖的重要病原之一,可感染多种虾种。罗氏沼虾(Macrobrachium rosenbergii)是中国养殖的重要沼虾品种之一。根据国际兽疫局(Office International des Epizooties,OIE)推荐的IHHNV检测方法,在中国大陆地区首次开展IHHNV在罗氏沼虾中的感染和流行情况调查。结果显示,在研究区的罗氏沼虾养殖区,IHHNV广泛流行,阳性率高达90%;但所有成年罗氏沼虾均未表现出明显的病症,仅表现为病毒的携带。通过基因序列分析显示,检测到的华南地区毒株属于Ⅰ型感染株,与菲律宾株进化关系较为接近;华东地区毒株属于Ⅱ型感染株,与东南亚株进化关系较近。本研究为IHHNV在罗氏沼虾内的感染、流行和防控提供了详细参考。
文摘A reverse transcription polymerase chain reaction (RT PCR) was used for detection of infectious hypodermal and haematopoietic necrosis virus (IHHNV) from the gills and subcutaneous tissue of Penaeus vannamei (10-15cm in body size) without clinical signs. With primers 77012R and 77353F, a fragment of 356bp of IHHNV was amplified in RT PCR. The amplified product was sequenced and showed more than 98% homologue with the sequences of other IHHNV strains in Gene Bank.
