武汉单极虫巢式聚合酶链反应检测方法的建立与优化  

Establishment of nested PCR assay for detection of Thelohanellus wuhanensis in allogynogenetic crucian carp

作　　者：柴下 陆宏达[1,2] 刘俊杰[1] 岳蒙蒙[1] 卢德刚 

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]上海海洋大学农业部淡水水产种质资源重点实验室,上海201306 [3]江苏省扬州市江都区水产管理站,江苏扬州225200

出　　处：《上海海洋大学学报》2014年第4期556-563,共8页Journal of Shanghai Ocean University

基　　金：上海高校知识服务平台(ZF1206);上海市重点学科建设项目(Y1101)

摘　　要：应用巢式聚合酶链反应(nested PCR)建立了一种检测异育银鲫武汉单极虫(Thelohanellus wuhanensis)基因组DNA的方法,提取的DNA作为模板进行两次扩增,第一次扩增用单极虫18S rRNA序列通用引物:5'-CTGCGGACGGCTCAG TAAATCAGT-3'和5'-CCAGGACATCTTAGGGCATCACAGA-3',扩增长度为1 584 bp;第二次依据第一次扩增产物中武汉单极虫特有的高度保守区设计特异性引物:5'-ACCCACTTCTGTGGC CTTTC-3'和5'-AATCCGACCTACAACGCTGG-3',扩增长度为853 bp。结果表明,通过PCR反应体系中退火温度、dNTP、Mg2+、延伸时间、扩增循环数和模板浓度的优化,优化后的巢式PCR最低DNA检测量达10 fg,优化后比常规PCR检测的灵敏度高104倍。因此,本文所建立的巢式PCR检测方法适合于水环境或水生动物中武汉单极虫的微量检测,也为异育银鲫武汉单极虫病的诊断和流行病调查提供了一种高灵敏的检测技术。The nested polymerase chain reaction （nested PCR） was developed to amplify a DNA segment of the Thelohanellus wuhanensis, which caused disease in allogynogenetic crucian carp [ Carassius auratus gibelio （ ♀ ） × Cyprinus carpio var singuonensis ♂ ） ]. The DNA of Thelohanellus wuhanensis was extracted as a template to be amplified. The first PCR amplification reacted with the primers ： 5 ＇- CTGCGGACGGCTCAGTAAATCAGT-3＇ and 5＇-CCAGGACATCTrAGGGCATCACAGA-3＇. The length of the first PCR amplified DNA was 1584 bp. The second nested PCR amplification reacted with the primers： 5＇- ACCCACTI＇CTGTGGCCTFFC-3＇ and 5＇-AATCCGAC CTACAACGCTGG-3＇. The length of the second nested PCR amplified DNA was 853 bp. After optimizing the annealing temperature, the time of extension, the amplification cycle number, the concentration of dNTP, magnesium and template, the DNA detected sensitivity of nested PCR was 10000 times higher than that of conventional PCR. The DNA detected quantity of nested PCR was only 10 fg. The established nested PCR could be used for the microquantity detection of the Thelohanellus wuhanensis and provided a more sensitive technique of disease diagnosis and molecular epidemiological survey.

关 键 词：异育银鲫 武汉单极虫 巢式PCR 检测方法 

分 类 号：S917[农业科学—水产科学]