Application and development of a TaqMan real-time PCR for detecting infectious spleen and kidney necrosis virus in Siniperca chuatsi.
Infectious spleen and kidney necrosis virus (ISKNV) is one of the major epidemiological agents that had caused great economic loss in Chinese perch (Siniperca chuatsi). In this study, a specific TaqMan real-time PCR was developed using a pair of primers and a TaqMan probe specific to the ORF007 gene of ISKNV to rapidly detect ISKNV copies in Chinese perch samples. This assay was optimized to produce linearity from 8.75 × 108to 8.75 × 101copies in standard curve with an efficiency of 98% and a R2value of 0.9999. Moreover, the minimum detection limit of this assay was 10,000 times more sensitive than that of conventional PCR method. The coefficients of variation of intra- and inter-assay repeatability were less than 2.4% and 3.3%, respectively. The viral distribution in different tissues of diseased Chinese perch was evaluated by TaqMan real-time PCR method and the highest level of viral copies was detected in spleen. Among the 76 diseased Chinese perch clinical samples, 35 and 29 were positive samples based on the TaqMan real-time PCR and conventional PCR methods, respectively, indicating that the TaqMan real-time PCR was more sensitive than conventional PCR. Therefore, the TaqMan real-time PCR should be a useful tool for the early surveillance and quantitation of ISKNV.
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou 510380, China.
Qiang
Q
Lin
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou 510380, China.
Xiaozhe
X
Fu
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou 510380, China.
Lihui
L
Liu
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou 510380, China.
Hongru
H
Liang
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou 510380, China.
Huizhi
H
Guo
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou 510380, China.
Shuwen
S
Yin
Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, College of Animal Sciences and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou, Guangdong, 510225, China.
Venkatesh
V
Kumaresan
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou 510380, China.
Zhibin
Z
Huang
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou 510380, China. Electronic address: liningq@126.com.
Ningqiu
N
Li
D000818:Animals; D002460:Cell Line; D002681:China; D017931:DNA Primers / Q000235:genetics; D004266:DNA Virus Infections / Q000175:diagnosis / Q000662:veterinary / Q000821:virology; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology; D007668:Kidney / Q000821:virology*; D009336:Necrosis / Q000821:virology*; D010473:Perciformes / Q000821:virology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013154:Spleen / Q000821:virology*
D017931:DNA Primers
Chinese perch; ISKNV; TaqMan real-time PCR