Li N, Xu B, Dong W, Qiao S, Lee L F, Zhang H M, Li M, Du N
College of Veterinary Medicine, China Agricultural University, Beijing 100094, China.
J Vet Med A Physiol Pathol Clin Med. 2007 Dec;54(10):553-8. doi: 10.1111/j.1439-0442.2007.01008.x.
Avian leukosis virus (ALV) subgroup J (ALV-J) is an exogenous ALV and causes myeloid leukosis in meat-type chickens. We have previously reported the isolation and identification of ALV-J in commercial layer flocks from 12 farms in northern China. In this report, we further characterized this virus by in situ polymerase chain reaction (PCR) hybridization in various affected organs of chickens from six of the 12 farms. A routine method for hybridization of nucleic acid uses radioactive probe, such as a P32-labelled probe. We found that the non-radioactive digoxigenin (DIG) probe is sensitive enough to detect the nucleic acid of virus in chicken tissues. We used a pair of published primers (H5/H7) specific to the gp85 envelope gene and 3' region of pol gene of prototype ALV-J strain HPRS-103. The total RNA extracted from tumour, bone marrow, oviduct, liver and spleen of the diseased chickens from six commercial flocks, and cDNA was successfully amplified. Using the primers and cDNA, we obtained an ALV-J-specific cDNA probe of 545 bp in length by PCR. In situ PCR with H5/H7 primers was carried out in the paraffin sections from tissues of the diseased chickens, followed by in situ hybridization using the DIG-labelled cDNA probe. Positive hybridization signals were detected in the cytoplasm of paraffin sections of tumours and other organ tissues. The intensity of the signals was documented using an image analysis system measuring integral optical density (IOD). The IOD values for tissue sections treated by in situ PCR hybridization are significantly higher than that by in situ hybridization alone (P < 0.01). These data taken together suggest that in situ PCR hybridization is a more sensitive technique for detection of ALV-J in tissue sections.
禽白血病病毒J亚群（ALV-J）是一种外源性禽白血病病毒，可导致肉用型鸡发生髓细胞性白血病。我们之前已报道了在中国北方12个养殖场的商品蛋鸡群中分离和鉴定出ALV-J。在本报告中，我们通过原位聚合酶链反应（PCR）杂交技术，对来自12个养殖场中6个养殖场的患病鸡的各种受影响器官中的这种病毒进行了进一步鉴定。核酸杂交的常规方法使用放射性探针，如P32标记的探针。我们发现，非放射性地高辛配基（DIG）探针足够灵敏，能够检测鸡组织中的病毒核酸。我们使用了一对已发表的引物（H5/H7），它们对原型ALV-J毒株HPRS-103的gp85包膜基因和pol基因的3'区域具有特异性。从6个商品鸡群的患病鸡的肿瘤、骨髓、输卵管、肝脏和脾脏中提取总RNA，并成功扩增出cDNA。使用这些引物和cDNA，我们通过PCR获得了一个长度为545 bp的ALV-J特异性cDNA探针。用H5/H7引物进行原位PCR，然后使用DIG标记的cDNA探针进行原位杂交，在患病鸡组织的石蜡切片中检测到阳性杂交信号。使用测量积分光密度（IOD）的图像分析系统记录信号强度。原位PCR杂交处理的组织切片的IOD值显著高于单独原位杂交处理的组织切片（P < 0.01）。综合这些数据表明，原位PCR杂交是检测组织切片中ALV-J的更灵敏技术。
