鸡传染性贫血病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立
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摘要
为建立鸡传染性贫血病毒(chicken infectious anemia virus,CIAV)的实时荧光定量PCR检测方法,本试验通过CIAV基因组保守区域设计1对扩增片段大小为180bp的特异性引物,构建pGM-T-CIAV重组质粒,制备阳性标准品,建立SYBR GreenⅠ实时荧光定量PCR标准曲线,并进行敏感性试验、特异性试验和重复性试验。结果显示,CIAV的Ct阈值与标准品浓度在5.33×108至5.33×103拷贝/μL间呈良好的线性关系,相关系数R2=0.998,斜率为-3.443,产物Tm值在86℃左右。该方法与网状内皮组织增生病病毒(REV)、禽白血病病毒(ALV)J亚型、马立克氏病病毒(MDV)、传染性法氏囊病病毒(IBDV)基因组均无交叉反应,敏感性为5.33拷贝/μL,比普通PCR高1 000倍,批内和批间重复试验变异系数均小于3%。结果表明,本试验建立的CIAV SYBR GreenⅠ实时荧光定量PCR检测方法可实现对鸡传染性贫血病的早期诊断及感染程度的定量分析的检测。
The aim of this study was to develop a Real-time PCR method for detection of chicken infectious anemia virus(CIAV).According to the CIAV genome conservative area,apair of specific primers which could amplify about 180 bp fragment was designed.By building the pGM-TCIAV recombinant plasmid,and preparing positive standard,the SYBR GreenⅠReal-time PCR standard curve was established.At the same time,sensitivity test,specificity test and repeatability test were determined.The results indicated that CIAV Ct threshold had good linear relationship with the standard concentration.The correlation rate and slope were 0.998and-3.443,respectively.The Tm was about 86 ℃.No cross-reactions were found between REV,ALV J subtype,MDV and IBDV.The sensibility was 5.33coppies/μL,which was 1 000 times than the traditional PCR.The coefficient of variation value was less than 3%.The CIAV SYBR Green Ⅰ Real-time PCR detection method could be realized on the early diagnosis of the disease and infection degreequantitative analysis of the test.
The aim of this study was to develop a Real-time PCR method for detection of chicken infectious anemia virus(CIAV).According to the CIAV genome conservative area,apair of specific primers which could amplify about 180 bp fragment was designed.By building the pGM-TCIAV recombinant plasmid,and preparing positive standard,the SYBR GreenⅠReal-time PCR standard curve was established.At the same time,sensitivity test,specificity test and repeatability test were determined.The results indicated that CIAV Ct threshold had good linear relationship with the standard concentration.The correlation rate and slope were 0.998and-3.443,respectively.The Tm was about 86 ℃.No cross-reactions were found between REV,ALV J subtype,MDV and IBDV.The sensibility was 5.33coppies/μL,which was 1 000 times than the traditional PCR.The coefficient of variation value was less than 3%.The CIAV SYBR Green Ⅰ Real-time PCR detection method could be realized on the early diagnosis of the disease and infection degreequantitative analysis of the test.
引文
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[6]黄恒,凌洪权,陈燕凌,等.传染性贫血病检测方法研究进展[J].湖北畜牧兽医,2013,34(1):82-84.
[7]王春梅,江波,李伟奇.鸡传染性贫血的研究[J].中国畜牧兽医,2009,36(11):139-140.
[8]童桂香,谢芝勋,刘加波,等.鸡传染性贫血病毒荧光定量PCR检测方法的建立[J].中国兽医学报,2009,29(6):696-699.
[9]王素华,王忠才,李孝军,等.牛副结核分枝杆菌实时荧光定量PCR检测方法的建立[J].中国畜牧兽医,2012,39(10):22-26.
[10]宋修庆,高宏雷,王晓艳,等.鸡贫血病毒TaqMan探针荧光定量PCR检测方法的建立[J].中国预防兽医学报,2009,31(1):56-59.
[11]周晓俊,朱淮民,邱璐.检测蚊虫感染黄病毒属病毒Real-time PCR方法的建立[J].中国病原生物学杂志,2008,3(8):561-563.
[12]Santhosh S R,Parida M M,Dash P K,et al.Development and evaluation of SYBR GreenⅠ-based one-step real-time RT-PCR assay for detection and quantitation of Japanese encephalitis virus[J].J Virol Methods,2007,143(1):73-80.
[2]赵锋,李学花,林世武,等.鸡传染性贫血病毒衣壳蛋白的原核表达及免疫原性分析[J].中国畜牧兽医,2013,40(8):34-38.
[3]张新珩,吴波良,陈峰,等.广东省鸡传染性病毒编码区基因的克隆与分子进化分析[J].中国兽医学报,2014,34(3):402-405.
[4]Fussell L W.Poultry industry strategies for control of immunosuppressive diseases[J].Poult Sci,1998,77(80):1193-1196.
[5]崔治中.免疫抑制性病毒多重感染在鸡群疫病发生和流行中的作用[J].中国家禽,2003,25(20):1-4.
[6]黄恒,凌洪权,陈燕凌,等.传染性贫血病检测方法研究进展[J].湖北畜牧兽医,2013,34(1):82-84.
[7]王春梅,江波,李伟奇.鸡传染性贫血的研究[J].中国畜牧兽医,2009,36(11):139-140.
[8]童桂香,谢芝勋,刘加波,等.鸡传染性贫血病毒荧光定量PCR检测方法的建立[J].中国兽医学报,2009,29(6):696-699.
[9]王素华,王忠才,李孝军,等.牛副结核分枝杆菌实时荧光定量PCR检测方法的建立[J].中国畜牧兽医,2012,39(10):22-26.
[10]宋修庆,高宏雷,王晓艳,等.鸡贫血病毒TaqMan探针荧光定量PCR检测方法的建立[J].中国预防兽医学报,2009,31(1):56-59.
[11]周晓俊,朱淮民,邱璐.检测蚊虫感染黄病毒属病毒Real-time PCR方法的建立[J].中国病原生物学杂志,2008,3(8):561-563.
[12]Santhosh S R,Parida M M,Dash P K,et al.Development and evaluation of SYBR GreenⅠ-based one-step real-time RT-PCR assay for detection and quantitation of Japanese encephalitis virus[J].J Virol Methods,2007,143(1):73-80.