中国畜牧兽医2016，Vol.43Issue(7)：1717-1722,6.DOI:10.16431/j.cnki.1671-7236.2016.07.009
猪伪狂犬病病毒SYBR GreenⅠ实时荧光定量PCR方法的建立
Establishment of SYBR GreenⅠReal-time Quantitative PCR for Detection Porcine Pseudorabies Virus
摘要
Abstract
In this study,a SYBR GreenⅠ dye based on Real-time quantitative PCR was established using the specific primers-pair according to gE gene characterization of porcine pseudorabies virus (PRV).The optimized results demonstrated that the detection assay with good linear determina-tion range from 7.53×101 to 7.53×106 copies per reaction.There was no cross-reaction occurred with nucleic acids extracted from the common porcine infectious diseases,such as porcine circovir-us 2 (PCV2),porcine parvovirus (PPV),Haemophilus parasuis and Streptococcus susi,no ampli-fication signals were detected.The results showed that the melting curve analysis with only one specific peaked with a melting temperature (Tm),which was (92.9±0.1)℃ at detecting PRV positive samples,also no specific peak could be detected for common porcine infectious diseases described above.Series experiments were carried out in order to assess the sensitivity,specificity and reproducibility for the method,following by the intra-assay and inter-assay CVs for Ct values obtained with the standard plasmids.The intra-assay and inter-assay statistics were 0.31% to 1.14% and 0.42% to 1.74%,respectively.All the results showed that the established method was sensitive,specific and reproducible,which meaned it could be used for the research of patho-genic mechanism of porcine pseudorabies virus.关键词
猪伪狂犬病毒/gE基因/SYBR GreenⅠ/实时荧光定量PCRKey words
porcine pseudorabies virus/gE gene/SYBR Green Ⅰ/Real-time quantitative PCR分类
畜牧业引用本文复制引用
陈如敬,吴学敏,陈秋勇,严山,车勇良,王晨燕,王隆柏,周伦江..猪伪狂犬病病毒SYBR GreenⅠ实时荧光定量PCR方法的建立[J].中国畜牧兽医,2016,43(7):1717-1722,6.基金项目
福建省属公益类科研专项(2014R1023-13、2015R1023-10) （2014R1023-13、2015R1023-10）
