畜牧与兽医2017，Vol.49Issue(6)：120-124,5.
内含NDV核酸的耐核糖核酸酶病毒样颗粒作为荧光定量RT-PCR内标的制备与鉴定
Preparation and identification of ribonuclease-resistant virus-like particles as an internal standard for Newcastle Real-time fluorescent RT-PCR
程帮照 1杨金兵 2卢运战 2诸亚平 2赵宏波 3刘鹏义3
作者信息
- 1. 宣城市畜牧兽医局,安徽宣城242000
- 2. 宣州区畜牧兽医管理局,安徽宣城242000
- 3. 安徽华卫集团禽业有限公司,安徽宣城242000
- 折叠
摘要
Abstract
This study aimed to prepare the internal control for detection of Newcastle disease virus (NDV) by fluorescent Real-time RTPCR.A pair of specific primers were designed to amplify the gene fragments of mature enzyme and capsid protein of phage MS2.The amplified fragments were inserted into plasmid pET32a to construct recombinant plasmid pET32a-MS2,and then identified by double enzyme digestion and PCR.Internal control fragment was prepared by chemical synthesis and Klenow enzyme linking technology according to the gene sequence used for RT-PCR detection of NDV,and inserted into the pET32a-MS2.The recombinant plasmid pET32a-MS2-IC was transformed into Escherichia coli BL21,and induced to express by IPTG.After the expression product was digested by RNase and DNase,ribonuclease-resistant virus-like particles was detected by fluorescence Real-time RT-PCR.The result indicated that the high expression level of virus-like particles was prepared as the internal control for Newcastle Real-time RT-PCR.关键词
新城疫/耐核糖核酸酶/病毒样颗粒/MS2噬菌体/荧光RT-PCRKey words
Newcastle disease/ribonuclease resistance/virus-like particles/phage MS2/Real-time RT-PCR分类
生物科学引用本文复制引用
程帮照,杨金兵,卢运战,诸亚平,赵宏波,刘鹏义..内含NDV核酸的耐核糖核酸酶病毒样颗粒作为荧光定量RT-PCR内标的制备与鉴定[J].畜牧与兽医,2017,49(6):120-124,5.
