Tong Guixiang, Wei Xinxian, Yin Weili, Liao Xiaoguang, Yang Kai, Fang Zhishan, Sun Tao, Yue Zhiqin, Li Xiaozheng
Guangxi Academy of Fishery Science, Nanning 530021, China.
Shandong Entry-Exit Inspection and Quarantine Bureau, Qingdao, Shandong 266000, China.
J AOAC Int. 2017 Jan 1;100(1):159-164. doi: 10.5740/jaoacint.16-0066.
A liquid chip technique was developed to detect spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV) of salmonids simultaneously. Sequences of the G gene of SVCV, N gene of IHNV, and G gene of VHSV were used to design SVCV-, IHNV-, and VHSV-specific primers, which were labeled with biotin and subjected to amination modification. They were then coupled with fluorescence-coded microspheres and used for hybridization with reverse-transcription PCR products of SVCV, IHNV, and VHSV. A BD FACSArray was used to detect fluorescence signal in the reaction system. This assay system had a high sensitivity to SVCV, VHSV, and IHNV, with LODs of 10, 10, and 100 pg/μL, respectively. Moreover, the assay was specific for the detection of SVCV, IHNV, and VHSV and was not susceptible to cross-detection of other viruses, including pike fry rhabdovirus, hirame rhabdovirus, infectious pancreatic necrosis virus, viral nervous necrosis virus, yellowtail ascites virus, grass carp reovirus, red sea bream iridovirus, and koi herpesvirus. The liquid chip assay technique established in this study provides a novel, convenient, and rapid approach for the detection of SVCV, IHNV, and VHSV.
开发了一种液相芯片技术，用于同时检测鲤春病毒血症病毒（SVCV）、传染性造血器官坏死病毒（IHNV）和鲑科鱼类的病毒性出血性败血症病毒（VHSV）。利用SVCV的G基因、IHNV的N基因和VHSV的G基因序列设计SVCV、IHNV和VHSV特异性引物，这些引物用生物素标记并进行胺化修饰。然后将它们与荧光编码微球偶联，并用于与SVCV、IHNV和VHSV的逆转录PCR产物杂交。使用BD FACSArray检测反应体系中的荧光信号。该检测系统对SVCV、VHSV和IHNV具有高灵敏度，检测限分别为10、10和100 pg/μL。此外，该检测方法对SVCV、IHNV和VHSV具有特异性，不易受到其他病毒的交叉检测影响，包括犬牙鳉弹状病毒、牙鲆弹状病毒、传染性胰脏坏死病毒、病毒性神经坏死病毒、黄尾腹水病毒草鱼呼肠孤病毒、真鲷虹彩病毒和锦鲤疱疹病毒。本研究建立的液相芯片检测技术为SVCV、IHNV和VHSV的检测提供了一种新颖、便捷、快速的方法。
