Department of Marine Biology and Aquaculture, College of Marine Science, Gyeongsang National University, 2, Tongyeonghaean-ro, Tongyeong 53064, Republic of Korea.
Int J Mol Sci. 2023 Feb 8;24(4):3426. doi: 10.3390/ijms24043426.
Red sea bream iridovirus (RSIV) is an important aquatic virus that causes high mortality in marine fish. RSIV infection mainly spreads through horizontal transmission via seawater, and its early detection could help prevent disease outbreaks. Although quantitative PCR (qPCR) is a sensitive and rapid method for detecting RSIV, it cannot differentiate between infectious and inactive viruses. Here, we aimed to develop a viability qPCR assay based on propidium monoazide (PMAxx), which is a photoactive dye that penetrates damaged viral particles and binds to viral DNA to prevent qPCR amplification, to distinguish between infectious and inactive viruses effectively. Our results demonstrated that PMAxx at 75 μM effectively inhibited the amplification of heat-inactivated RSIV in viability qPCR, allowing the discrimination of inactive and infectious RSIV. Furthermore, the PMAxx-based viability qPCR assay selectively detected the infectious RSIV in seawater more efficiently than the conventional qPCR and cell culture methods. The reported viability qPCR method will help prevent the overestimation of red sea bream iridoviral disease caused by RSIV. Furthermore, this non-invasive method will aid in establishing a disease prediction system and in epidemiological analysis using seawater.
真鲷虹彩病毒（RSIV）是一种重要的水生病毒，可导致海水鱼类高死亡率。RSIV 感染主要通过海水水平传播，早期检测有助于预防疾病爆发。虽然实时荧光定量 PCR（qPCR）是一种检测 RSIV 的敏感和快速方法，但它无法区分感染性和非感染性病毒。在这里，我们旨在开发一种基于吖啶橙（PMAxx）的存活 qPCR 检测方法，吖啶橙是一种光活性染料，可穿透受损的病毒颗粒并与病毒 DNA 结合，以防止 qPCR 扩增，从而有效区分感染性和非感染性病毒。我们的结果表明，75μM 的 PMAxx 可有效抑制存活 qPCR 中热灭活 RSIV 的扩增，从而区分非感染性和感染性 RSIV。此外，与传统 qPCR 和细胞培养方法相比，基于 PMAxx 的存活 qPCR 检测方法更有效地选择性检测海水中的感染性 RSIV。该方法将有助于预防因 RSIV 引起的真鲷虹彩病毒病的高估。此外，这种非侵入性方法将有助于使用海水建立疾病预测系统和进行流行病学分析。
