摘要
采用PCR方法克隆了猪圆环病毒2型（PCV2）ORF2基因，将构建的重组质粒pMD-ORF2作为PCV2实时荧光定量PCR（FQ-PCR）检测的标准阳性模板，并对该模板进行了酶切及测序鉴定。结果显示，此模板特异性强，稳定性好，一20℃保存20d无显著变化；对起始浓度为1．0×10^6、1．0×10^5、1．0×10^4拷贝／弘L的pMD-ORF2标准品的最终实际测得值分别为1．000×10^4、0．935×10^5和0．987×10^4，其变异系数分别为2．527％、2．067％和2．092％。表明，以此模板进行FQ—PCR具有良好的准确性和重现性。该模板的检测线性范围宽，当稀释度为1．0×10^9～1．0×10^5拷贝／μL时，相关系数r=0．989。
An open reading frame（ORF） 2 gene of porcine circovirus（PCV） serotype 2 was amplified by PCR and cloned into pMD-18 T vector to construct a recombinant plasmid pMD-ORF2 to be used as standard positive template for the real-time fluorescent quantitative PCR（FQ-PCR） for detection of PCV2. The pMD-ORF2 was identified by digestion with Kpn Ⅰ Hind Ⅲ and sequencing. The pMD-ORF2 was high specific and stable for the FQ-PCR. The final values on initiative concentrations of 1. 0 × 10^6, 1.0×10^5, 1.0 ×10^4copies/μL of DNA were 1. 000×10^6, 0. 935×10^5 and 0. 987×10^4 and their coefficients of variation were 2. 527%,2. 067% and 2. 092% ,respectively. The results demonstrated that the plasmid for FQ-PCR was favourable accurate and repeatable,with wide linear range. The correlation coefficient was -0. 989 when dilution was among 1.0×10^9 to 1.0×10^5 copies/μL.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2007年第1期49-53,共5页
Chinese Veterinary Science
基金
国家高技术研究发展计划(863)项目(2003AA241110)
广东省农业科技攻关项目(2003B21404和20052490100)
关键词
猪圆环病毒2型
实时荧光定量PCR
模板
porcine circovirus serotype 2
real time fluorescent quantitative PCR
template
