title: Reverse transcription loop-mediated isothermal amplification for rapid and quantitative assay of covert mortality nodavirus in shrimp.
abstract: A disease known as covert mortality disease has become an increasing problem in the shrimp farming industry in recent years in China and several countries of Southeast Asia, leading to serious losses in production. Litopenaeus vannamei (also known as Pacific white shrimp) is affected by this disease that leads to a range of clinical symptoms including hepatopancreas atrophy and necrosis, soft shell, slow growth, and abdominal muscle whitening and necrosis in the acute stage of disease. A new nodavirus, termed covert mortality nodavirus (CMNV), has been shown to be the etiological agent. In this study, we report a sensitive and specific real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and quantitative detection of CMNV. The optimal conditions for this newly developed RT-LAMP reaction were found to be 6mM MgCl2and 1.6mM dNTPs, an incubation temperature of 65°C and a reaction time of 50min. The analytical sensitivity of the RT-LAMP assay was estimated to be 6.3pg total RNA of CMNV-infected shrimp and 27 copies of the target plasmid. The diagnostic sensitivity and specificity of the newly developed assay versus the standard nested reverse transcription PCR (RT-PCR) assay was 96.4% and 94.4%, respectively. The reaction products were detected by visual inspection after staining with an in-tube DNA fluorescent dye, a measure taken to eliminate the risk of contamination. The quantitative RT-LAMP assay for CMNV showed high correlation coefficient (r2=0.9953) when the initial templates were above 1000 copies, however the correlation coefficient decreased when the initial templates were lower than 1000 copies. Test of viral load in shrimp indicated that the viral loads varied from 1.5×102to 6.7×106copies per mg of cephalothorax tissue. Thus, the CMNV RT-LAMP assay is a sensitive and specific new tool for the field detection and quantification of CMNV in the diagnosis and surveillance of covert mortality disease.
authors.affiliation: Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute (YSFRI), Chinese Academy of Fishery Sciences (CAFS), Qingdao 266071, China; National Laboratory for Marine Science and Technology, Qingdao 266071, China; Shanghai Ocean University, Shanghai 201306, China. Electronic address: zhangql@live.com.
authors.forename: Qingli
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute (YSFRI), Chinese Academy of Fishery Sciences (CAFS), Qingdao 266071, China; Shanghai Ocean University, Shanghai 201306, China. Electronic address: liushuang8@163.com.
authors.forename: Shuang
authors.initials: S
authors.lastname: Liu
authors.affiliation: Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute (YSFRI), Chinese Academy of Fishery Sciences (CAFS), Qingdao 266071, China; Shanghai Ocean University, Shanghai 201306, China. Electronic address: yanghlwin@163.com.
authors.forename: Haolin
authors.initials: H
authors.lastname: Yang
authors.affiliation: Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute (YSFRI), Chinese Academy of Fishery Sciences (CAFS), Qingdao 266071, China; Shanghai Ocean University, Shanghai 201306, China. Electronic address: zhuluoluo666@163.com.
authors.forename: Luoluo
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute (YSFRI), Chinese Academy of Fishery Sciences (CAFS), Qingdao 266071, China; National Laboratory for Marine Science and Technology, Qingdao 266071, China. Electronic address: wanxy@ysfri.ac.cn.
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Wan
authors.affiliation: Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute (YSFRI), Chinese Academy of Fishery Sciences (CAFS), Qingdao 266071, China; Shanghai Ocean University, Shanghai 201306, China.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute (YSFRI), Chinese Academy of Fishery Sciences (CAFS), Qingdao 266071, China; National Laboratory for Marine Science and Technology, Qingdao 266071, China; Shanghai Ocean University, Shanghai 201306, China. Electronic address: huangjie@ysfri.ac.cn.
authors.forename: Jie
authors.initials: J
authors.lastname: Huang
mesh_terms: D000818:Animals; D030381:Nodaviridae / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques; D033561:Penaeidae / Q000821:virology*; D012327:RNA Virus Infections / Q000821:virology*; D048348:Reverse Transcription; D019562:Viral Load
keywords: Covert mortality nodavirus; Litopenaeus vannamei; RT-PCR; Reverse transcription loop-mediated isothermal amplification; Shrimp