鲑鱼甲病毒阳性核酸物质制备及RT-PCR检测方法的建立  

Positive nucleic acid material preparation and establishment of a RTPCR for salmonid alphavirus

作　　者：刘敏[1] 杜航[1] 宋傲臣 高帅[1] 唐丽杰[2] 蒋烨[1] 李一经[2] 

机构地区：[1]东北农业大学动物科学与技术学院,哈尔滨150030 [2]东北农业大学动物医学学院,哈尔滨150030

出　　处：《东北农业大学学报》2016年第7期56-62,共7页Journal of Northeast Agricultural University

基　　金：国家科技支撑计划项目(2013BAD12B02)

摘　　要：为建立快速检测鲑鱼甲病毒(Salmonid alphavirus,SAV)RT-PCR方法,根据Gen Bank公布的E2基因序列(1 375 bp)设计引物,扩增S AV E2全长基因,利用假病毒制备技术获得包裹E2核酸物质(RNA)的SAV假病毒溶液。以SAV假病毒作为阳性核酸物质质控品,以E2F:5'CCG-TTG-CGG-CCA-CAC-TGG-ATG 3',E2R:5'CCT-CAT-AGG-TGA-TCG-ACG-GCA-G 3'为引物,优化反应条件,建立SAV的RT-PCR检测方法。结果表明,该方法可扩增SAV E2特异性516 bp的DNA片段,引物最适反应浓度为1.0μmol·L-1,最佳退火温度为57.5℃,对SAV的三种亚型SAV1(V4640)、SAV2(V4619)、SAV5(V4638)检测结果均为阳性,对SVCV、IHNV和IPNV的PCR扩增结果均为阴性;并确定对SAV的核酸最低检出量达1.59 pg;以RT-PCR方法在不同时间对每份样品作3次重复检测,检测结果一致。表明此方法特异性强,敏感性高,稳定性和重复性较好,可用于SAV临床诊断和检测。To rapidly identify the pathogens causing salmonid alphavirus （SAV）, a RT-PCR was developed with primers designed according to the sequences of the E2 gene （1 375 bp） of SAV available in GenBank. Amplified the SAV E2 gene, using false virus preparation technology to obtain the SAV fake virus solution wrapped with E2 material nucleic acid （RNA）. SAV RT-PCR detection method was established with the SAV false virus as positive nucleic acid material quality control and the designed primers of E2F： 5＇ CCG-TTG-CGG CCA-CAC-TGG-ATG 3＇, E2R： 5＇ CCT-CATOAGG TGA-TCG-ACG-GCA-G 3＇ as detection primers. Results showed that we could amplify the specific fragment of SAV E2 DNA gene （516 bp） better when the optimum concentration of primers was 1.0 IJmol- L1 and the annealing temperature was 57.5 ℃ at the same time. Detection result of SAV1, SAV3, SAV5 were all positive by the established RT-PCR method, but no specific amplifications from other virus（SVCV, IPNV, IHNV）, this assay could detecting the minimum detection of SAV nucleic acids was 1.59 pg. Each sample had been detectived three times at different times through this RT-PCR, and there was no change between the detection results of same samples.Comprehensive research results showed the established RT-PCR （use the SAV false virus nucleic acid material as positive control） had strong specificity, high sensitivity, good stability and repeatability characteristics, could be used in clinical diagnosis and detection of SAV.

关 键 词：鲑鱼甲病毒 SAV E2 核酸物质 RT-PCR 检测方法 

分 类 号：S942.3[农业科学—水产养殖]