title: Establishment and application of cross-priming isothermal amplification coupled with lateral flow dipstick (CPA-LFD) for rapid and specific detection of red-spotted grouper nervous necrosis virus.
abstract: BACKGROUND
  Red-spotted grouper nervous necrosis virus (RGNNV) is an important pathogen that causes diseases in many species of fish in marine aquaculture. The larvae and juveniles are more easily infected by RGNNV and the cumulative mortality is as high as 100 % after being infected with RGNNV. This virus imposes a serious threat to aquaculture of grouper fry. This study aimed to establish a simple, accurate and highly sensitive method for rapid detection of RGNNV on the spot.
  
  
  METHODS
  In this study, the primers specifically targeting RGNNV were designed and cross-priming isothermal amplification (CPA) system was established. The product amplified by CPA was detected through visualization with lateral flow dipstick (LFD). Three important parameters, including the amplification temperature, the concentration of dNTPs and the concentration of Mg(2+) for the CPA system, were optimized. The sensitivity and specificity of this method for RGNNV were tested and compared with those of the conventional RT-PCR and real-time quantitative RT-PCR (qRT-PCR).
  
  
  RESULTS
  The optimized conditions for the CPA amplification system were determined as follows: the optimal amplification temperature, the optimized concentration of dNTPs and the concentration for Mg(2+) were 69 °C, 1.2 mmol/L and 5 mmol/L, respectively. The lowest limit of detection (LLOD) of this method for RGNNV was 10(1) copies/μL of RNA sample, which was 10 times lower than that of conventional RT-PCR and comparable to that of RT-qPCR. This method was specific for RGNNV in combination with SJNNV and had no cross-reactions with 8 types of virus and bacterial strains tested. This method was successfully applied to detect RGNNV in fish samples.
  
  
  CONCLUSIONS
  This study established a CPA-LFD method for detection of RGNNV. This method is simple and rapid with high sensitivity and good specificity and can be widely applied for rapid detection of this virus on the spot.
authors.affiliation: Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, P. R. China, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (CAFS), Qingdao, P. R. China. suzidan08@163.com.|College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, P. R. China. suzidan08@163.com.
authors.forename: Zi Dan
authors.initials: ZD
authors.lastname: Su
authors.affiliation: Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, P. R. China, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (CAFS), Qingdao, P. R. China. shicy@ysfri.ac.cn.|Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, P. R. China. shicy@ysfri.ac.cn.
authors.forename: Cheng Yin
authors.initials: CY
authors.lastname: Shi
authors.affiliation: Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, P. R. China, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (CAFS), Qingdao, P. R. China. huangjie@ysfri.ac.cn.|Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, P. R. China. huangjie@ysfri.ac.cn.
authors.forename: Jie
authors.initials: J
authors.lastname: Huang
authors.affiliation: Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, P. R. China, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (CAFS), Qingdao, P. R. China. gmshen1989@126.com.|College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, P. R. China. gmshen1989@126.com.
authors.forename: Gui Ming
authors.initials: GM
authors.lastname: Shen
authors.affiliation: Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, P. R. China, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (CAFS), Qingdao, P. R. China. lj311@126.com.
authors.forename: Jin
authors.initials: J
authors.lastname: Li
authors.affiliation: Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, P. R. China, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (CAFS), Qingdao, P. R. China. wsq098886@163.com.|College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, P. R. China. wsq098886@163.com.
authors.forename: Sheng Qiang
authors.initials: SQ
authors.lastname: Wang
authors.affiliation: Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, P. R. China, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (CAFS), Qingdao, P. R. China. fanchao1126@126.com.|College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, P. R. China. fanchao1126@126.com.
authors.forename: Chao
authors.initials: C
authors.lastname: Fan
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D005393:Fish Diseases / Q000175:diagnosis*; D005399:Fishes; D030381:Nodaviridae / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D019095:Point-of-Care Systems; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
chemical_list: D017931:DNA Primers