摘要
为了建立一种特异、灵敏、快速、重复性好和检测成本低的羊口疮病毒检测方法,试验根据GenBank中羊口疮病毒(登录号为AY386264)ORFVgORF102基因DNA序列,应用Beacon Designer7.0软件设计、合成了1对SYBR GreenⅠ实时荧光定量PCR引物。结果表明:经反应条件优化后,建立了羊口疮病毒SYBR GreenⅠ实时荧光定量PCR,最佳引物浓度为400 nmol/L,最佳模板浓度为3.61～36.1 ng/μL或2.385×107～2.385×108copies/μL;该方法组内及组间变异系数均小于2%,检测灵敏度可达到3.61 pg/μL或2.385×104copies/μL,上机检测时间不超过1 h;运用该方法对2份临床样品进行定量检测,样品抽提DNA中病毒拷贝数分别为1.4×109copies/μL和8.7×108copies/μL。说明试验所建立的羊口疮病毒SYBR Green I实时荧光定量PCR具有特异、灵敏、快速、重复性好和检测成本低等优点,适合于羊口疮病毒临床样品的检测。
To establish a detection method for sheep off virus, which is sensitive, specific, fast, repeatable and low -cost, a pair of specific primers for SYBR Green I real - time PCR was designed by Beacon Designer software （ version 7.0） according to ORFVgORF102 gene DNA sequence of sheep off virus genome（ Accession： AY386264） published on GenBank. The results showed that the SYBR Green I real - time PCR detection method for sheep off virus was established after the reaction conditions were optimized. The optimal concentration of the primers and templates were 400 nmol/L and 3.61-36.1 ng/μL or 2. 385×10^7- 2. 385×10^8 copies/μL, respectively. The coefficients of variation of intra - and inter - assay for the assay were both less than 2%. The detection sensitivity reached 3.61 pg/μL or 2. 385 × 10^4 copies/μL, and the detection time was less than one hour. The assay was used for quantitatively detecting two clinical samples of sheep off virus that prevailed in Yunnan. The virus contents in extracted DNA from the specimens were 1.4 ×10^9copies/μL and 8.7×10^8 copies/μL, respectively. The results indicate that the established assay has the advantages of being sensitive, specific, fast, repeatable and low - cost, and is suited for the detection of sheep off virus in clinical samples.
出处
《黑龙江畜牧兽医》
CAS
北大核心
2013年第5期15-19,22,共6页
Heilongjiang Animal Science And veterinary Medicine
基金
云南省科技厅农业科技创新工程项目(2008LA019)
