中国水产科学2011，Vol.18Issue(3)：661-666,6.DOI:10.3724/SP.J.1118.2011.00661
蛙虹彩病毒巢式PCR检测方法的建立
Development of a nested PCR detection method for Ranavirus
摘要
Abstract
In this study, a nested PCR for detecting soft-shelled turtle iridovirus (STIV), Epizootic haematopoietic necrosis virus (EHNV) and Tiger frog virus (TFV) was developed.Two pairs of primers were designed based on the sequence of major capsid proteins (MCP) gene of STIV by bioinformatics software DNAStar and primer primer 5.The outer primers were P1 (5'-CGCATGTCTTCTGTAAC TGG-3') and P2 (5'-CGTTACAAG ATTGGGAATCC-3').The inner primers were P3 (5'-CGCGATAGGCTACTAT AACATGG -3') and P4 (5'- AGA TGTGTGACGTTCTGCACC -3').The first round PCR was carried out using PCR Supermix with 40 ng template DNA, 0.6 μL primers (P1/P2, 50 pmol/μL), 2 μL PCR buffer with MgCl2, 0.5 μL Tag, and dH2O was added to reach total volum of 20 μL.The conditions of PCR were: 94℃ for 3 min, then 94℃ for 30 s, 50℃ for 30 s, 72℃ for 1 min, 30 cycles, followed by an extension reaction at 72℃ for 10 min.The nested PCR was carried out by using the same supermix except 0.5 μL template DNA, the product of first round PCR, 0.6 μL primers (P3/P4, 50 pmol/μL).The conditions were the same with the first round PCR.In this study, a standard recombinant plasmid was produced to in nested PCR assay.The DNA products of first round PCR were extracted, and then cloned into pGem-T Easy vector.After sequencing, the recombinant plasmids named pGem-T-S were used in ranavirus detection as the position control.Specificity and sensitivity of the nested PCR method were estimated.Data showed that the detection limit of this assay was 102 copies for standard recombinant plasmid.It was also found that the specificity of this assay was high without any cross-reactions with DNA from Infectious haematopoietic necrosis (IHNV), Spring viraemia of carp virus (SVCV), Viral Haemorrhagic Septicaemia virus (VHSV), Channel catfish virus (CCV), Infectious pancreatic necrosis (IPNV), Red sea bream iridovirus (RSIV), Hirame rhabdovirus (HRV), Koi Herpesvirus (KHV) and Infectious spleen and kidney necrosis virus (ISKNV) provided by Shenzhen Exit-entry Inspection and Quarantine Bureau.Only STIV, EHNV and TFV DNA could result in the same pattern of curves with the two primers.In summary, all data confirmed that the nested PCR assay is a powerful tool for the detection of ranavirus with high sensitivity and specificity.Moreover, the nested PCR is of low cost and time saving in contrast to ELISA assay, indirect fluorescent antibody tests and immunoelectron microscopy.So the Nest-PCR method could be used to detect these three kinds of ranavirus efficiently.关键词
蛙虹彩病毒/中华鳖虹彩病毒/流行性造血器官坏死病毒/虎纹蛙虹彩病毒/巢式PCRKey words
Ranavirus/ soft-shelled turtle iridovirus/ epizootic haematopoietic necrosis virus/ tiger frog virus/ nested PCR分类
农业科技引用本文复制引用
张旻,林祥梅,江育林..蛙虹彩病毒巢式PCR检测方法的建立[J].中国水产科学,2011,18(3):661-666,6.基金项目
国家质量监督检验检疫总局科技项目(2008IK010) （2008IK010）
中国检验检疫科学研究院(CAIQ)基础科研业务经费项目(2008JK004). （CAIQ）
