PCV-2d基因型的PCR-RFLP诊断方法的建立  

Establishment of PCR-RFLP differential diagnosis method for PCV-2d genotype

作　　者：郑庆礼 吴昊宸 黄瑞玲 许文煌 张誓育 王全溪[1] ZHENG Qingli;WU Haochen;HUANG Ruiling;XU Wenhuang;ZHANG Shiyu;WANG Quanxi(College of Animal Science,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,Chin)

机构地区：[1]福建农林大学动物科学学院,福建福州350002

出　　处：《福建农林大学学报（自然科学版）》2018年第4期446-451,共6页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：福建农林大学大学生创新性项目(201710339194);福建农林大学科学发展基金(KF2015094)

摘　　要：猪圆环病毒2型(porcine circovirus type 2,PCV2)有多个基因型,其中基因型d的感染率仅次于基因型b,建立PCV-2d基因型的PCR-RFLP诊断方法具有重要意义.根据NCBI中PCV2 Cap基因序列,设计1对特异性引物,并选择PCV-2d特异的酶切位点MSPI进行酶切,对酶切的体系和条件进行优化,同时进行敏感性、重复性试验.结果表明:PCR总体积25μL,模板量2μL,退火58℃,35个循环为PCR反应最佳条件,目的片段702 bp;MSPI酶能将PCV2d特异地切出2个条带474、228 bp.酶切体系为12.5μL、MSPI酶1μL、酶切时间1 h;该酶切体系的最低模板量为10 ng·μL-1,重复性好.送检的24份病料的PCV2d阳性率为33.3%.可见,本研究建立的PCV-2d基因型的PCR-RFLP鉴别诊断方法,特异、敏感.Porcine circovirus type 2 has many genetic types,in which the infection level of type d is only less than b type. So it is important to establish the PCR-RFLP differential diagnosis of PCV-2 d genotypes. According to the sequence of PCV2 cap gene in NCBI,a pair of specific primers was designed. The PCR reaction system and reaction conditions were optimized,and the specificity test was carried out. The specific restriction site of PCV-2 d was MSPI enzymes. The enzyme digestion system and conditions were optimized,and the sensitivity and repeated enzyme digestion experiments were carried out simultaneously. The results showed that the target fragment could be amplified specifically,and the length was about 702 bp. PCV-2 d can be cut into 474 bp and 228 bp two fragments. The total volume was 12.5 μL,including the enzyme amount 1 μL,and the enzyme digestion time was 1 h. The sensitivity test showed that the minimum template amount of the enzyme digestion system was 10 ng · μL-1. The results of repeated tests showed that the enzyme digestion system can be repeated. The positive rate of PCV-2 d in 24 samples was 33. 3%. Therefore,the PCR-RFLP differential diagnosis of PCV-2 d genotypes was specific and sensitive.

关 键 词：猪圆环病毒2型 基因型 限制性片段长度多态性聚合酶链反应 

分 类 号：S852.651[农业科学—基础兽医学]