口蹄疫O、Asia 1分型彩色胶乳试纸条诊断方法的建立

Development of a Rapid Colour Latex Particles Immunochromatographic Strip Test for the Diagnosis of Foot-and-Mouth Disease Virus O and Asia 1 Type

为建立一种快速、简便、直观的口蹄疫O、Asia 1分型彩色胶乳免疫层析的方法,作者选择2种不同颜色的单分散胶乳,将纯化的Asia 1/China/05流行毒株兔抗体制备蓝色免疫胶乳;将纯化的O/China/99流行毒株兔抗体制备红色免疫胶乳。将2种免疫胶乳分别喷涂于不同玻璃纤维上,晾干叠加制成彩色胶乳反应垫。再分别将纯化的Asia 1/YN/BSh/58/B和AV99(L)标准毒株的豚鼠抗体固定于同一硝酸纤维素膜的不同区域上作为2条检测带。最后组装成口蹄疫O、Asia 1分型彩色胶乳试纸条。通过对阳性参考样品的检测,结果显示试纸条有很好的灵敏度,可检测到病毒含量为3.90×104 LD50;在检测与口蹄疫临床症状相似病原,如猪水泡病病毒(SVDV)、水泡性口炎(VS)、水泡性疹(VES)抗原时无交叉反应,特异性好;不同批次间、同一批次内的试纸条,在检测阴、阳性参考样品时,结果完全一致,说明试纸条重复性好;试纸条对检测已知血清型的74份田间样品的符合性试验中,O型、Asia 1型样品的阳性符合率分别为95.24%、96.30%,阴性样品符合率为100%。本试验研发的口蹄疫O、Asia 1分型彩色胶乳试纸条具有很好的灵敏度、特异性、重复性和符合性,而且可以通过不同颜色进行定型检测,适合国内流行的口蹄疫病原型别的分型检测。

To develop a sensitive,rapid and simple colour latex particles immunochromatography assay for determination of foot-and-mouth disease virus（FMDV） O,Asia 1 type from the field samples,Mondispersed polystyrene Microspheres with two distinct dyes and carboxyl group in the size rang of 380 nm were prepared with seed polymerization and swelling.The purified anti-FMDV strain Asia 1/China 05,O/China99 rabbit antibody were coupled with blue and red latex particles.The purified anti-FMDV Asia 1,O type guinea pig antibodies were wrapped on to nitrocellulose membrane as two test line.The FMDV serotype diagnosis kit was then performed and conditions were determined.The seriate results indicated that sensitivity of the test kit reached 3.90×104LD50.No cross reaction was found with SVD,VS,VES antigen by cross tests.In the clinic test,a total 74 field samples were detected with the test list.The corresponding rate between the strip test kit and traditional methods of positive samples for FMDV Asia 1,O type and negative samples were 96.30%,95.24%,100%,respectively.In this study,the established color latex particles immunochromatographic strip test kit is sensitive,specific for detecting FMDV Asia 1,O type and is potentially useful for the high throughput screening,the test for the diagnosis of food-and-mouth disease virus O and Asia 1 type.