检测猪瘟病毒野毒株胶体金免疫层析方法的建立  

Development of a colloidal gold immunochromatographic assay for antigen detection of wild-type classical swine fever virus

作　　者：王向鹏[1,2] 孙元[1] 杨增岐[2] 仇华吉[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/猪传染病研究室,黑龙江哈尔滨150001 [2]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《中国预防兽医学报》2010年第6期441-445,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家科技支撑计划(2006BAD06A03)

摘　　要：为建立快速、简便检测猪瘟病毒(CSFV)野毒的胶体金免疫层析方法(GICA),本研究采用柠檬酸三钠还原法制备胶体金颗粒,标记纯化的抗CSFVE2蛋白的单克隆抗体(MAb)6E10作为捕捉抗体,将纯化的抗CSFVE2蛋白的MAbHQ06和兔抗鼠IgG抗体包被在硝酸纤维素膜上,分别作为检测线和质控线,优化反应条件,组装成胶体金免疫层析试纸条。结果表明,所制备的试纸条用于检测CSFV野毒感染的PK-15细胞培养物,在检测线和质控线处均呈现红色条带,健康PK-15细胞培养物对照仅在质控线呈现红色条带;试纸条检出病毒培养物的最低限为103.5 TCID50;用不同批次的试纸条重复检测,结果无差异;该试纸条不与猪瘟兔化弱毒(HCLV)、牛病毒性腹泻病毒(BVDV)、猪繁殖与呼吸综合征病毒(PRRSV)、传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)、猪轮状病毒(PRV)、伪狂犬病病毒(PrV)、猪细小病毒(PPV)和猪圆环病毒2型(PCV2)反应。所制备的试纸条具有良好的特异性、敏感性和重复性,初步达到了区分检测CSFV野毒株和弱毒株的目的。A colloidal gold immunochromatographic strip was developed for the rapid detection of the wild-wpe classical swine fever virus （CSFV） antigens using two MAb against the E2 protein. The wild-type CSFV-specific 6E10 MAb was labeled with colloidal gold as capture antibody and the HQ06 MAb was immobilized on the test line as detection antibody, while a rabbit anti-mouse IgG antibody was blotted on the control line of the nitrocellulose membrane. The strips were tested on CSFV infected PK-15 cell cultures and normal cells. The results showed that specific signal developed in the test line when detecting the CSFV-infected cells, but not with the non-infected cells. The detection limit of the strip was 10 3. 5 TCID50 CSFV. The test strips did not react with hog cholera lapinized virus, bovine viral diarrhea virus porcine reproductive and respiratory sypdrome virus, transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine rotavirus, pseudorabies virus, porcine parvovirus, and porcine circovirus type 2. Taken together, the test strip was specific, sensitive and reproducible, which could be used to differentiate cells infected with wild-type CSFV from those infected with HCLV.

关 键 词：猪瘟病毒 野毒株 胶体金 免疫层析 

分 类 号：S852.65[农业科学—基础兽医学]