【作者】 王峰； 刘青； 王晓伟； 陈洪博； 于琳琳； 王玥； 姜艳萍； 成子强；

【Author】 WANG feng,LIU qing,WANG Xiao-wei,CHEN Hong-bo,YU Lin-lin,WANG Yue,CHENG Zi-qiang~* (College of Animal Science & Veterinary Medicine,Shandong Agricultural University, Tai’an,Shandong 271018,China)

【机构】 山东农业大学动物科技学院；

【摘要】 将ALV-J NX0101毒株接种1日龄和7日龄SPF雏鸡并设阴性对照组,采用实时荧光定量逆转录PCR方法,定期检测病毒在体内的复制情况.根据Genebank发表的ALV-J env基因保守序列(AY897227)设计一对特异性引物扩增目的基因;根据鸡的3-磷酸甘油醛脱氢酶(GAPDH)基因序列(K01458),在保守区内设计一对引物扩增内参基因,分别转入质粒作为标准品制作标准曲线,采用SYBR GreenⅠ染料建立荧光定量PCR法,并对方法的特异性,敏感性,重复性进行评价.结果表明:标准曲线的Ct值与标准品浓度的对数值之间存在线性关系;最低可检测到60个拷贝/反应的病毒数,比常规PCR灵敏度高1000倍.检测结果分别采用绝对定量法和相对定量法进行分析,均达到了良好的效果.通过对病毒含量变化的检测发现,在雏鸡4周龄时,两个接毒组ALV-J病毒突然呈对数式增长.据此分析ALV-J病毒在体内经过3-4周的潜伏后,突然呈爆发式增长,这种情况可能和临床表现的免疫抑制直接相关;结果还显示,7日龄接毒比1日龄的病毒扩增更加显著.本研究建立了一种特异性强、敏感性高、可定量分析ALV-J病毒增殖的方法,为进一步相关研究奠定了基础.更多还原

【Abstract】 In order to detect the change of avian leukosis virus subgroup J(ALV-J) in SPF chickens that were inoculated with ALV-NX0101 virus separately at 1 day and 7 days.A pair of primers was designed to target conservative of env gene(AY897227) as the target gene,another pair of primers was designed to target conservative of GAPDH gene of chicken(K01458) as the internal control gene.The standard curve was constructed by using positive plasmids.The specificity,sensitivity and reproducibility of the assay were evaluated.The results showed:there was a broad linear range between Ct value and logarithm of plasmid concentration;the detection limit was 60 copies / reaction for positive standard sample,1000-folds more sensitive when compared with the conventional PCR using the same primers.There were both good detective results that were analyzed separately by absolute quantification and relative quantification.The quantum of virus was logarithmic growth after 3-4 weeks that the chickens were inoculated with virus.It probable was related to clinical situation of immune suppression.Furthermore,the virus amplification of that was inoculated at 1 day was more patency than 7 days.This study developed a specific,sensitive, quantificational method for detecting ALV-J.更多还原