摘要：建立了美人鱼发光杆菌杀鱼亚种的环介导等温扩增技术(Loop-Mediated Isothermal Amplification,LAMP)。针对IGS2基因保守序列的6个区域设计2对特异引物,利用链置换DNA聚合酶(Bst DNA polymerase)在恒温65℃下保温40 min。采用钙黄绿素作为显色剂,FTA卡采集病样肝组织中病原菌核酸。结果显示:以IGS2基因建立的LAMP方法能够特异性检测美人鱼发光杆菌杀鱼亚种,检测灵敏度为2.6×102 cfu/mL。针对美人鱼发光杆菌杀鱼亚种IGS2基因建立的LAMP检测方法具有较好的特异性和稳定性,可用于美人鱼发光杆菌杀鱼亚种的野外现场快速检测。

摘要：以cfb基因保守序列的6个区域设计4条特异性引物,利用链置换DNA聚合酶（Bst DNA polymerase）在恒温（65℃）下保温40 min,建立了无乳链球菌Streptococcus agalactiae的LAMP技术。建立的LAMP方法能够特异性地检测无乳链球菌,检测灵敏度为3.7×10^1～3.7×10^2cfu/mL,反应前加入显色剂钙黄绿素避免二次污染。现场应用中采用FTA卡采集红尾皇冠鱼Aequidens rivulatus病鱼肝脏组织病原菌,操作简便、快捷。研究表明,针对无乳链球菌cfb基因建立的LAMP检测方法具有较高的特异性及稳定性,尤其是具有快速、简便、成本低等优点,可用于无乳链球菌的野外现场快速检测。

摘要：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.

摘要：本研究建立了一种检测对虾传染性皮下及造血组织坏死病毒(Infectious hypodermal and hematopoietic necrosis virus, IHHNV)的环介导等温扩增法(Loop-mediated isothermal amplification, LAMP)。根据GenBank中注册的IHHNV-VN基因序列设计出6条特异性引物,成功建立了IHHNV-LAMP检测方法。该方法检出限为6 copies/μl,且与对虾其它常见疫病均无交叉反应。采用LAMP和常规PCR方法对40份具有典型IHHNV感染症状的对虾样品进行检测,结果显示:LAMP方法的阳性检出率为97.5%,PCR方法的阳性检出率为85%,表明建立的IHHNV-LAMP方法不仅具有高灵敏度、高特异性而且还有较高的准确性,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。

摘要：一、概况天津市宁河县天河养殖场有养殖水面270亩,其中:主养团头鲂水面160亩,设计团头鲂产量600公斤/亩左右,套养少量鲫鱼和鲢鳙鱼.