基于牡蛎疱疹病毒DNA聚合酶基因的巢式PCR检测方法的建立及应用  

The development and application of nested PCR detection method for Ostreid herpesvirus-1 based on DNA polymerase gene

作　　者：高文辉[1,2] 白昌明[2,3] 蔡生力[1] 王崇明[2,3] 

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]中国水产科学研究院黄海水产研究所,农业部海洋渔业可持续发展重点实验室,山东青岛266071 [3]青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室,山东青岛266071

出　　处：《水产学报》2016年第3期326-333,共8页Journal of Fisheries of China

基　　金：国家自然科学基金(31502208);现代农业产业技术体系建设专项(CARS-48);青岛海洋科学与技术国家实验室鳌山科技创新计划(2015ASKJ01)~~

摘　　要：为了建立适用于Os HV-1不同变异株的检测方法,在牡蛎疱疹病毒(Os HV-1)3个变异株全基因组序列比对的基础上,筛选到牡蛎疱疹病毒基因组中高度保守的DNA聚合酶(DNA polymerase)基因,据此设计巢式PCR引物,优化PCR反应体系和条件,建立了基于Os HV-1 DNA聚合酶基因的巢氏PCR检测方法(P-n PCR检测方法),利用P-n PCR与Cn PCR检测方法对不同年份和宿主来源的Os HV-1疑似感染样本进行检测。结果显示,Pn PCR检测方法能稳定地检出100拷贝/μL的病毒DNA;P-n PCR较C-n PCR检测方法具有更强的特异性和更高的检出率。研究表明,本研究建立的P-n PCR检测方法适用于Os HV-1不同变异株的检测,可为该病毒的检测和流行病学调查提供可靠的技术支持。In order to establish a detection method suitable for different strains of Os HV-1,the primers were designed based on the nuclei acid sequence of the conserved DNA polymerase of Os HV-1. A nested PCR detection method（P-n PCR） was established by optimization of the annealing temperatures of the primers and protocols of PCR. Then, both P-n PCR and C-n PCR were employed to test the infection status of the samples collected from different years and hosts. Our results indicated that the detection limits of the P-n PCR detection method was about 100 copies/μL of recombinant plasmid containing Os HV-1 genes. P-n PCR was more specific than C-n PCR in the detection of different variants of Os HV-1, and resulted in a higher prevalence of Os HV-1 for the same samples. In conclusion, a P-n PCR detection method was developed to detect different variants of Os HV-1infection. The high specificity of P-n PCR to Os HV-1 ensured that different variants of Os HV-1 could be detected as early as possible, which will provide reliable technical support for the detection and epidemiology studies of Os HV-1.

关 键 词：牡蛎 疱疹病毒 巢式PCR 分子变异 特异性 

分 类 号：S944[农业科学—水产养殖]