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当前位置: 首页 > 文章 > 羊口疮病毒SYBRGreenI染料法实睚荧光定量PCR的建立及运用 黑龙江畜牧兽医 2013 (5) 15-19,22
Position: Home > Articles > Establishment and application of SYBR Green I real -time fluorescence  quantitative PCR for sheep off virus Heilongjiang Animal Science and Veterinary Medicine 2013 (5) 15-19,22

羊口疮病毒SYBRGreenI染料法实睚荧光定量PCR的建立及运用

作  者：
姚俊;彭海生;鲁富有;张乔明;李富祥;李华春
关键词：
羊口疮病毒;SYBR;Green;I;实时荧光定量PCR;建立;应用
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12疮病毒检测方法,试验根据GenBank中羊口疮病毒(登录号为AY386264)ORFVgORFl02基因DNA序列,应用BeaconDesigner7.O软件设计、合成了1对SYBRGreenI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SYBRGreenI实时荧光定量PCR,最佳引物浓度为400nmol/L,最佳模板浓度为3.61-36.1ng/μL或2.385*10^7-2.385*10^8copies/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3.61pg/μL或2.385*10^4copies/IxL,上机检测时间不超过1h;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*10^9copies/μL和8.7*10^8copies/μL.说明试验所建立的羊12疮病毒SYBRGreenI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.
译  名：
Establishment and application of SYBR Green I real -time fluorescence  quantitative PCR for sheep off virus
关键词：
sheep off virus%SYBR Green I% real - time fluorescence quantitative PCR% establishment% application
摘  要：
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.

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