禽白血病J亚群病毒实时荧光定量PCR检测方法的建立及应用

摘要

The organs distributions of Avian Leukosis Virus subgroup J (ALV-J) in chicken were studied through the method of real-time PCR in this article. A SYBR Green I-based real-time PCR was developed for detection of ALV-J by using a pair of primers which was designed to target NX0101 (5 258-5 510 bp). The product of PCR (253 bp) was linked to pMD18-T vector served as standard curve. The sensitivity, specificity and repeatability tests of the method were conducted. The hearts, livers, spleens, lungs, kidneys, thymuses, bursa of Fabricius and glandular stomachs of 20 chickens were detected repeatedly 3 times by this method. The results showed a precise linear relationship with a correlation coefficient of R2 =0. 991 4; the detection limits was 81 copies of DNA plasmid, it was 100 times more sensitive than RT-PCR. The meltingrn curve showed a single peak could only be detected for ALV-J. The variation coefficient of repeatability tests were less than 5%. The copies of ALV-J in thymuses were highest and lungs, spleen, and bursa of Fabricius were higher, and hearts were lowest. Furthermore, the ALV-J copies in natural infection chickens were higher than artificial infection chickens. Real-time PCR about ALV-J gene established in this article was high sensitivity, strong specificity and short testing period. The organs distributions of ALV-J in chicken were preliminarily discussed. The research provided important technical support for the diagnosis and treatment with ALV-J.%本研究旨在通过荧光定量PCR方法研究禽白血病J亚群病毒(ALV-J)在家禽体内各个器官的分布.根据ALV-J NX0101毒株基因5 258- 5 510 bp的碱基序列,设计了1对特异性引物,进行RT-PCR反应,扩增产物为253 bp,将该产物连接T载体作为Real-time PCR反应的标准品,利用SYBR Green Ⅰ染料进行Real-time PCR反应,建立了标准曲线,并进行反应灵敏性,特异性和重复性试验.然后用已建立的方法对人工感染ALV-J的SPF鸡心脏、肝脏、脾脏、肺脏、肾脏、胸腺、法氏囊、腺胃进行3次重复检测,将Ct值带入标准曲线公式得出各个组织的病毒拷贝数.试验结果表明:标准曲线线性关系R值均为0.991 4,检测极限约为81拷贝质粒DNA,比RT-PCR灵敏100倍以上；特异性分析表明只有ALV-J能检测到特异性的熔解度峰值；批内和批间重复性试验的变异系数均小于5％.通过比较数值以得出胸腺ALV-J基因含量是最高的,肺脏、脾脏、法氏囊含量也比较高,心脏拷贝数最低.自然感染发病鸡各器官ALV-J基因均高于人工感染ALV-J的基因含量.本研究所建立的ALV-J基因实时荧光定量RT-PCR方法灵敏度高、特异性强、检测周期短,初步探讨了禽白血病J亚群病毒在畜禽体内各个器官的病毒分布.这为以后禽白血病的诊断以及治疗提供了重要的技术支持.