水产学报2013，Vol.37Issue(2)：281-287,7.DOI:10.3724/SP.J.1231.2013.38214
栉孔扇贝急性病毒性坏死病毒巢式PCR检测方法的建立
Establishment of a nested PCR for rapid detection of Chlamys farreri acute viral necrosis virus
摘要
Abstract
The scallop Chlamys farreri is one of the major species cultured in North China, and its culture in commercial scale has been performed for more than 20 years. However, the great expansion and intensification have induced the occurrence of disease called " acute viral necrosis disease" ( AVND) since 1990's,and the cumulative mortality could be higher than 90%. This disease is caused by a virus called "acute viral necrosis virus"(AVNV),which is a spherical enveloped virus(130 to 170 nm in diameter) with spike-like surface protrusions and has been becoming the major limiting factor in the development of the scallop industry. In order to establish a rapid diagnosis method of AVNV parasitizing on scallop( C. farreri) , a pair of primers of nested-PCR were developed by Accelrys gene 2. 5 based on the conserved region of the AVNV genome ( GenBank accession number; GQ153938 ) in this study. The reactive conditions such as concentration of Mg2 + , dNTPs and annealing temperature were optimized for the PCR system, and the expected products of the external and internal primers were 979 and 548 bp respectively. The results showed that the primers were specific for AVNV and did not amplify marine aquaculture animals' s and bacteria' s genome DNA,and the method can be stably amplified 5 x 10 copies virus particles in 5 pg total nucleic acid of scallop tissue. Also this method was successfully applied to the Sanggou Bay scallop samples detected. Therefore, it is confirmed that the method will be very useful for sensitive and specific detection of AVNV in the laboratory, and has high specificity, good reproducibility, which will definitely facilitate the monitoring of the epidemic disease in the future.关键词
栉孔扇贝/急性病毒性坏死病毒/巢式PCRKey words
Chlamys farreri/acute viral necrosis virus (AVNV)/nested PCR分类
农业科技引用本文复制引用
李晨,王崇明,曲朋,黄倢..栉孔扇贝急性病毒性坏死病毒巢式PCR检测方法的建立[J].水产学报,2013,37(2):281-287,7.基金项目
现代农业产业技术体系建设专项(CARS-48) （CARS-48）
科技部国际合作项目(2010DFA31720) （2010DFA31720）
