Key Laboratory of Tropical Aquatic Germplasm of Hainan province, Sanya Oceanographic Institution, Ocean University of China, Sanya 572024, China; MOE Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Qingdao 266003, China.
Key Laboratory of Tropical Aquatic Germplasm of Hainan province, Sanya Oceanographic Institution, Ocean University of China, Sanya 572024, China; MOE Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Qingdao 266003, China.
J Invertebr Pathol. 2024 Jul;205:108143. doi: 10.1016/j.jip.2024.108143. Epub 2024 May 27.
Infectious myonecrosis virus (IMNV) has affected shrimp farming in many countries, such as northeastern Brazil and southeast Asia, and poses a serious threat to the global shrimp industry. Reverse transcription enzymatic recombinant amplification technology (RT-ERA) is a rapid DNA amplification assay with high specificity in isothermal conditions and has been widely applied to the pathogen's detection. In this study, two novel ERA assays of IMNV, real-time RT-ERA and an RT-ERA combined with lateral flow dipsticks assay (RT-ERA-LFD), were developed and evaluated. The real-time RT-ERA assay could be carried out at 38-42 °C and had the highest end-point fluorescence value and the smallest Ct value at 41 °C. The brightness and width of the detection line were at a maximum at 39 °C and 30 min, and these conditions were selected in RT-ERA-LFD. Both real-time RT-ERA and RT-ERA-LFD produced positive results with IMNV standard plasmids only and showed no cross-reaction with Vibrio parahaemolyticus, which causes acute hepatopancreatic necrosis disease (VpAHPND); white spot syndrome virus (WSSV); infectious hypodermal and hematopoietic necrosis virus (IHHNV); or Ecytonucleospora hepatopenaei (EHP). Meanwhile, we compared the sensitivities of nested RT-PCR, real-time RT-PCR, real-time RT-ERA, and RT-ERA-LFD. The sensitivities of real-time RT-ERA and RT-ERA-LFD were both 10 copies/μL. The detection sensitivities of nested RT-PCR and real-time RT-PCR were 10 and 10 copies/μL, respectively. As a result, two ERA assays were determined to be specific, sensitive, and economical methods for the on-site diagnosis of IMNV infection, showing great potential for the control of IMNV infections.
传染性肌肉坏死病毒（IMNV）已对巴西东北部和东南亚等许多国家的虾类养殖业造成影响，对全球虾类产业构成严重威胁。逆转录酶重组扩增技术（RT-ERA）是一种在等温条件下具有高特异性的快速 DNA 扩增检测方法，已广泛应用于病原体检测。本研究中，我们开发并评估了两种新型的 IMNV ERA 检测方法，即实时 RT-ERA 和 RT-ERA 与侧向流动条带检测（RT-ERA-LFD）的组合。实时 RT-ERA 检测可在 38-42°C 下进行，在 41°C 时具有最高的终点荧光值和最小的 Ct 值。检测线的亮度和宽度在 39°C 时达到最大值，且在 30 分钟时达到最大宽度，这些条件被选用于 RT-ERA-LFD。实时 RT-ERA 和 RT-ERA-LFD 仅对 IMNV 标准质粒产生阳性结果，与引起急性肝胰腺坏死病（VpAHPND）的副溶血弧菌、白斑综合征病毒（WSSV）、传染性皮下和造血坏死病毒（IHHNV）或对虾血细胞虹彩病毒（EHP）均无交叉反应。同时，我们比较了巢式 RT-PCR、实时 RT-PCR、实时 RT-ERA 和 RT-ERA-LFD 的灵敏度。实时 RT-ERA 和 RT-ERA-LFD 的灵敏度均为 10 拷贝/μL。巢式 RT-PCR 和实时 RT-PCR 的检测灵敏度分别为 10 和 10 拷贝/μL。因此，这两种 ERA 检测方法被确定为用于现场诊断 IMNV 感染的特异性、灵敏性和经济的方法，在控制 IMNV 感染方面具有很大的潜力。
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