J亚群禽白血病SYBR GreenⅠ荧光定量PCR检测方法的建立  

Development of SYBR GreenⅠreal-time RT-PCR assay for the detection of subgroup J avian leukosis virus

作　　者：王莉[1] 黄安宁[1] 李槿年[1] 张丹俊[2] 刘雪兰[1] 

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036 [2]安徽省农业科学院畜牧兽医研究所,安徽合肥230031

出　　处：《中国预防兽医学报》2013年第9期733-737,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：安徽省科技攻关战略性新兴产业项目(11010302119);国家自然基金项目(31272696)

摘　　要：为建立一种能够快速、灵敏和特异地检测J亚群禽白血病病毒(ALV-J)的SYBR GreenⅠ荧光定量PCR方法,本研究以ALV-J原型病毒株HPRS-103的pol基因3'端与gp85编码基因之间的保守区域(5 258 bp～5 802 bp)为检测目的片段,构建重组质粒并作为靶基因,通过对其浓度、引物浓度和退火温度的优化,建立了ALV-J SYBR GreenⅠ荧光定量PCR方法。结果显示该方法的最低检测限度为2.36×102拷贝/μL,比普通PCR高100倍;与其他禽源病毒无交叉反应;批内和批间变异系数均小于5%。表明本研究建立的方法具有良好的特异性、稳定性和灵敏性。采用该方法对100只临床病鸡进行检测,ALV的检出率为44%。随机选择10只感染阳性鸡,检测病毒基因在心脏、肝脏、脾脏、肺脏和肾脏中的分布与表达水平,结果表明ALV-J在各主要脏器中均有分布,但以肾脏中的病毒表达量最高。In this study, a SYBR GreenⅠ-based real-time RT-PCR assay for detecting avian leukosis virus subgroup J （ALV-J） was developed. The conservative region （5,258 nt to 5,802 nt） between pol and gp85 gene of the HPRS-103 strain of ALV-J was amplified by RT-PCR and cloned into pMD18-T to serve as positive template to establish the real-time PCR standard curve. The results indicated that the assay was highly sensitive with a detection limit of 2,360 copies for the positive recombinant plasmid, which was 100 time sensitive than the conventional PCR, and was highly specific without cross-reaction to other avian viruses. The coefficient of variation for intra- and inter-assay was both less than 5%. A total of 44 out of 100 clinical suspected disease chickens （44%） were positive for ALV-J. Moreover, ten of the ALV-J positive chicks were further detected in various organ tissues which shown the ALV-J genes were found in all organs detected and the kidney had the highest viral gene load. These data demonstrated that the SYBR GreenⅠ-based real-time RT-PCR could be as a routine assay for the clinical diagnosis of ALV-J infection.

关 键 词：J亚群禽白血病病毒 SYBR Green Ⅰ 荧光定量PCR 病毒基因表达水平 

分 类 号：S852.65[农业科学—基础兽医学]