Li Jiahao, Wu Huiliang, Xu Wei, Wang Yajun, Wang Hao, Wang Yingying, Li Yingying, Shi Cunbin, Bergmann Sven M, Mo Xubing, Wang Qing, Yin Jiyuan
Key Laboratory of Fishery Drug Development of Ministry of Agriculture and Rural Affairs, Key Laboratory of Aquatic Animal Immune Technology of Guangdong Province, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, Guangdong, PR China.
College of Veterinary Medicine, South China Agricultural University, China.
J Virol Methods. 2023 Feb;312:114663. doi: 10.1016/j.jviromet.2022.114663. Epub 2022 Nov 28.
Hemorrhagic disease of grass carp, which is induced by grass carp reovirus II (GCRV-II), leads to mass mortality in grass carp culture and causes enormous economic loss. However, there is currently no quantitative analysis method for the detection of GCRV-II, which is greatly restricted the etiological and epidemiological study of the disease. In this study a reverse transcription TaqMan PCR (RT-qPCR) assay was developed for the quantitative detection of GCRV-II. The probe and primers targeted location is the segment 6 (S6) region of the GCRV-II genome which is highly conserved. Standard curves were drawn and criteria were confirmed after the determination of the optimum reaction conditions. The species-specific assay showed that the method is highly specific and has no cross reactions with other pathogens. The assay was sufficiently sensitive to detect as low as 10 copies of virus RNA. Moreover, the method has a very good repeatability for batches and inter-batches sample detection. Then the method was applied to detect the virus in tissue samples from clinically infected grass carp, compared with conventional RT-seminested PCR, the RT-qPCR represents a specific value for detection rate of positive samples. In summary, the RT-qPCR was applied and achieved high sensitivity and specificity for GCRV-II detection.
草鱼出血病由草鱼呼肠孤病毒II型（GCRV-II）引起，导致草鱼养殖中大量死亡并造成巨大经济损失。然而，目前尚无用于检测GCRV-II的定量分析方法，这极大地限制了该疾病的病因学和流行病学研究。在本研究中，开发了一种逆转录TaqMan PCR（RT-qPCR）检测方法用于GCRV-II的定量检测。探针和引物靶向的位置是GCRV-II基因组的第6节段（S6）区域，该区域高度保守。在确定最佳反应条件后绘制标准曲线并确定标准。种属特异性检测表明该方法具有高度特异性，与其他病原体无交叉反应。该检测方法灵敏度足够高，可检测低至10个病毒RNA拷贝。此外，该方法在批次和批间样品检测中具有非常好的重复性。然后将该方法应用于检测临床感染草鱼的组织样品中的病毒，与传统的RT-半巢式PCR相比，RT-qPCR在阳性样品检测率方面具有特定价值。总之，RT-qPCR被应用并在GCRV-II检测中实现了高灵敏度和特异性。
