Key Laboratory of Healthy Freshwater Aquaculture, Ministry of Agriculture, Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, China; Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, China.
Key Laboratory of Healthy Freshwater Aquaculture, Ministry of Agriculture, Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, China.
J Invertebr Pathol. 2023 Jun;198:107921. doi: 10.1016/j.jip.2023.107921. Epub 2023 Apr 5.
Nucleic acid sequence-based amplification (NASBA) provides a fast and convenient approach for nucleic acid amplification under isothermal conditions, and its combination with an immunoassay-based lateral flow dipstick (LFD) could produce a higher detection efficiency for M. rosenbergii nodavirus isolated from China (MrNV-chin). In this study, two specific primers and a labelled probe of the capsid protein gene of MrNV-chin were constructed. The process of this assay mainly included a single-step amplification at a temperature of 41 ℃ for 90 min, and hybridization with an FITC-labeled probe for 5 min, with the hybridization been required for visual identification during LFD assay. The test results indicated that, the NASBA-LFD assay showed sensitivity for 1.0 fg M. rosenbergii total RNA with MrNV-chin infection, which was 10 times that of the present RT-PCR approach for the detection of MrNV. In addition, no products were created for shrimps with infection of other kinds of either DNA or RNA virus, which indicated that the NASBA-LFD was specific for MrNV. Therefore, the combination of NASBA and LFD is a new alternative detection method for MrNV which is rapid, accurate, sensitive and specific without expensive equipment and specialised personnel. Early detection of this infectious disease among aquatic organisms will help implement efficient therapeutic strategy to prevent its spread, enhance animal health and limit loss of aquatic breeds in the event of an outbreak.
核酸序列扩增（NASBA）在等温条件下提供了一种快速便捷的核酸扩增方法，其与基于免疫测定的横向流动试纸条（LFD）的结合可以提高对中国分离的罗氏沼虾诺达病毒（MrNV-chin）的检测效率。在本研究中，构建了 MrNV-chin 衣壳蛋白基因的两个特异性引物和一个标记探针。该检测方法的主要过程包括在 41℃温度下进行 90 分钟的单步扩增，以及与 FITC 标记探针杂交 5 分钟，在 LFD 检测中需要杂交才能进行目视识别。试验结果表明，NASBA-LFD 检测法对感染 MrNV-chin 的 1.0 fg 罗氏沼蟹总 RNA 具有敏感性，比目前 RT-PCR 检测 MrNV 的方法高 10 倍。此外，对于感染其他 DNA 或 RNA 病毒的虾没有产生产物，表明 NASBA-LFD 对 MrNV 具有特异性。因此，NASBA 和 LFD 的结合是一种新的替代检测 MrNV 的方法，具有快速、准确、敏感和特异性，不需要昂贵的设备和专业人员。早期检测水生生物中的这种传染病有助于实施有效的治疗策略以防止其传播，增强动物健康并限制在爆发时水生品种的损失。
