原位PCR和原位杂交检测蛋鸡J亚群禽白血病病毒

Detection of Avain Leukosis Virus Subgroup J in Egg-type Chickens Using In situ PCR and In situ Hybridization

根据ALV_J原型株HPRS10 3株gp85基因的内部序列 ,和pol基因的 3′端设计一对引物H5 H7。从发生ML病死蛋用型鸡的肿瘤、骨髓、肝脏、脾脏和输卵管组织中提取总RNA ,反转录为cDNA ,经PCR扩增得到长度为 5 4 5bp的ALV_JcDNA特异性探针。探针定位于 5 2 5 8～ 5 80 2bp。将病鸡的组织石蜡切片置HybaidExpress原位PCR仪平台上 ,以H5 H7为引物进行原位PCR扩增。应用地高辛标记的cDNA探针对原位PCR扩增后切片进行了原位杂交检测。结果在待检组织肿瘤组织、十二指肠、骨髓中出现明显的阳性信号。睾丸、肺、胰腺、大脑、输卵管、肾脏均检出散在的阳性信号。这是国内外首次从分子水平证明蛋鸡J亚群禽白血病。

A pair of primers H5/H7 was designed according to the sequence of encoding gene for partial gp85 encoded gene and 3′ region of pol gene of prototype ALV-J strain HPRS-103. A 545bp special probe of ALV cDNA, located in the 5258～5802 was obtained by amplifying of total RNA extracted from the tumor, bone marrow,oviduct, liver, and spleen from the diseased chickens with various flocks and was transformed reversely to cDNA. In situ PCR with H5/H7 primers was carried out in the paraffin section of diseased chickens, then in situ hybridization was carried out with the digoxigenin-labeled special cDNA probe over the amplified paraffin section. Marked positive signals were present in tumor, duodenum, bone marrow, and widespread positive signals were detected in the testis, lung, pancreas, cerebrum, oviduct, kidney. This is the first confirmed at the molecular level that avian leukosis subgroup J occurred in the egg-type chickens in the world.