【摘要】 为探索核酸杂交技术在测定J亚群禽白血病病毒(ALV-J)细胞半数感染量(TCID50)中的可行性,将ALV-J传染性克隆r NX0101株以10倍梯度稀释后按常规方法接种已长满单层鸡胚成纤维细胞(CEF)的96孔板,在维持7 d后收集细胞培养上清。上清液部分按照试剂盒说明书进行p27抗原的ELISA检测,另一部分提取RNA后以鸡致病性外源性禽白血病病毒特异性核酸探针交叉斑点杂交检测试剂盒进行Dot-blot检测,每孔中细胞固定后以ALV-J特异性单克隆抗体进行IFA检测,确定病毒感染孔后按照Reed-Muench法分别计算三种方法测定的TCID50。结果显示ELISA法、IFA法和Dot-blot法在确定病毒感染中能够相互验证和补充,该病毒在CEF的TCID50分别为10-4.7TCID50/0.1 m L、10-5.2TCID50/0.1 m L和10-5.3TCID50/0.1 m L。表明采用Dot-blot法测定ALV-J的TCID50是可行的,不仅能够排除内源性的干扰,并且比ELISA和IFA具有更高的灵敏度。更多还原

【Abstract】 To use Dot-blot in the test of TCID50 of avian leukosis virus subgroup J(ALV-J),one ALV-J stock r NX0101 was diluted from 10-1to 10-8and inoculated into CEF cells culture in 96-well plates,each dilution was inoculated into 8 wells in the same row and the cell plate was cultured with additional 7 days. The supernatant of each well was divided into two parts,half was submitted to ELISA test for p27 antigen and half was detected by Dot-blot with ALV specific probe. The cell was detected with IFA to insure the amplification of ALV-J. The TCID50 was calculated in CEF and there were 10-4.7TCID50/0.1 m L,10-5.2TCID50/0.1 m L and 10-5.3TCID50/0.1 m L by ELISA,IFA and Dot-blot respectively. The study confirmed that Dot-blot was more sensitive than IFA and ELISA in the detection of ALV-J and it could be used in the test of TCID50 of ALV-J in CEF.更多还原