高致病性猪蓝耳病免疫金标试纸条的研制与应用

The development and application of gold immunochromatography assay for highly pathogenic PRRSV

本试验利用柠檬酸三钠还原法制备粒径约为40 nm的胶体金,在玻璃纤维和硝酸纤维素膜的检测线和质控线上分别喷上胶体金PRRSV多克隆抗体、HP-PRRSV单克隆抗体和羊抗鼠IgG二抗。本试验组装的免疫胶体金诊断试纸条具备良好的敏感性和较高的特异性,最低病毒检测限为100TCID50;只对高致病性猪蓝耳病呈阳性反应,对PRRSV(ATCC VR2332)有弱反应,对猪瘟病毒、猪传染性胃肠炎病毒、猪伪狂犬病毒、猪圆环病毒均无反应。重复性试验表明,所研制的试纸条结果重复性较强,4℃条件下至少可保存半年。应用该试纸条对60份临床样品检测,同时与高致病性蓝耳病RT—PCR诊断试剂盒的方法进行符合性比较验证,结果显示两种方法检测结果的符合率达91%(10/11),证明本研究制备的高致病性猪蓝耳病抗原免疫金标试纸条是快速便捷的检测高致病性猪蓝耳病的新型检测技术。

The colloidal gold particles of about 40 nm in diameter were prepared by trisodium citrate reduction method. The detection line in glass fiber and quality control line in nitrocellulose membrane was respectively sprayed with colloidal gold PRRSV antibodies, HP PRRSV monoclonal antibody and sheep anti mouse IgG. The minimum detection limit of virus of the strips assembled by this test was 100TCID50 with a good sensitivity. The strips had high specificity, it could only detect the highly pathogenic PRRSV and, the PRRSV （ATCC VR2332） had a weak response. CSFV, TGEV, PRV, and PCV were all negative. The repeated experiments showed that the test strips had good repeatability. The test strips could be stored at 4℃ for at least six months. The strips were applied in 60 clinical sample testing, and at the same time the highly pathogenic PRRSV RT--PCR diagnostic kits methods were conducted for comparison and verification. The results showed that the conformity of the testing results of two methods was 91% （ 10/11 ） , suggesting that the test were new detection technology strips for quick and easy detection of the highly pathogenic PRRSV.