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当前位置: 首页 > 文章 > 栉孔扇贝急性病毒性坏死病毒巢式PCR检测方法的建立 水产学报 2013,37 (2) 124-130
Position: Home > Articles > Establishment of a nested PCR for rapid detection of Chlamys farreri acute viral necrosis virus Journal of Fisheries of China 2013,37 (2) 124-130

栉孔扇贝急性病毒性坏死病毒巢式PCR检测方法的建立

作  者：
李晨;王崇明;曲朋;黄倢
单  位：
中国水产科学研究院黄海水产研究所
关键词：
栉孔扇贝;急性病毒性坏死病毒;巢式PCR
摘  要：
为更好地实现对养殖海区栉孔扇贝急性病毒性坏死病毒(acute viral necrosis virus,AVNV)的快速诊断和分子流行病学的调查,以及AVNV的疫情监测,选择AVNV全基因组序列中的保守区段,应用Accelrys gene 2.5软件设计一对巢式引物,用于AVNV的检测。结果显示,引物的扩增片段分别为979和548 bp。实验优化了PCR体系中Mg2+和dNTPs浓度及扩增程序中的退火温度,并建立了完善的AVNV巢式PCR检测技术。研究表明,该PCR检测技术具有较高的敏感性,可稳定检测出5 pg扇贝样品组织总核酸中5×10 copies的病毒粒子。
译  名：
Establishment of a nested PCR for rapid detection of Chlamys farreri acute viral necrosis virus
作  者：
LI Chen1,WANG Chongming1,QU Peng1,2,HUANG Jie1(1.Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071,China; 2.Key Laboratory of Mariculture,Ministry of Education,Ocean University of China,Qingdao 266003,China)
关键词：
Chlamys farreri;acute viral necrosis virus(AVNV);nested PCR
摘  要：
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 5×10 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.

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