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

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 510bp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253bp,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。

Development and Application of Real-time PCR for Detection of Avian Leukosis Virus Subgroup J

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 gland-ular 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 melting 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.