口蹄疫病毒O、A、AsiaⅠ型定型诊断胶体金免疫层析方法的建立

Development of a Rapid Gold Immunochromatographic Strip Test for the Diagnosis of Foot-and-Mouth Disease Virus O,A and AsiaⅠ Type

【目的】建立一种从田间样品中快速、灵敏、简便检测O、A、AsiaI型口蹄疫病毒抗原的胶体金免疫层析方法(GICA)。【方法】采用柠檬酸三钠还原制备胶体金颗粒,标记纯化的标准株O、A、AsiaI型口蹄疫抗体。将纯化的口蹄疫流行株O/China99,A/AF72and AsiaⅠ/China05抗体和羊抗豚鼠抗体分别包被在硝酸纤维素膜上,作为检测带与质控带。经试验条件优化,组装形成胶体金定型诊断试剂盒。【结果】本试验研制的口蹄疫定型试剂盒具有良好的灵敏度,可检测到7.8×104LD50(1﹕128倍稀释乳鼠毒)的病毒量。同时对阳性及阴性样品进行3次重复检测结果完全相同,说明其有较好的重复性;特异性试验证实该方法在检测口蹄疫临床症状相似病原,如猪水疱病抗原(SVD)、水泡性口炎(VS)、水泡性疹(VES)等病毒时无交叉反应;试剂盒对206份已知血清型田间及实验室样品的符合率试验,O、A、AsiaⅠ型和阴性样本的符合率分别为92.45%、91.66%、92.75%、100%定型准确率94.58%。试剂盒在4℃下可保存6个月。【结论】本试验研发的口蹄疫病毒胶体金定型诊断试剂盒是一种快速、灵敏、特异的FMD抗原检测方法,对基层现场具有广泛应用价值。

[ Objective ] To develop a sensitive, rapid and simple gold immunochromatography assay （G1CA） for determination of foot-and-mouth disease virus （FMDV） O, A, Asia I type from the field samples. [Method] The purified anti-FMDV O, A, Asia I type Guinea pig antibody were coupled with the colloidal gold, and it was obtained by reducing the gold chloride with sodium citrate. The purified anti-FMDV strain O/China99, A/AF72 and Asia I/China05 rabbit antibody and the goat anti- Guinea pig IgG were wrapped onto nitrocellulose membrane as the test line （T line） and the control line （C line）. The FMDV serotyping diagnosis kit was then performed by further optimization of the labeling and the conditions were determined. [Result]The seriate results indicated that the sensitivity of the test kit reached 7.8×10^4 LD50. （1 ： 128 dilution mouse tissue virus） and it had the same results for positive and negative specimens tested in three times. No cross reaction was found with Swine vesicular disease （SVD）, VS, VES antigen by cross tests. In the clinic test, a total of 206 field samples were comparatively detected with GICA. The corresponding rate of gold immunochromatographic strip test kit for FMDV O, A, Asia I type and negative sample was 92.45%, 91.66%, 92.75% and 100%. The correct rate of detected FMDV serotype was 94.58%. This strip could be stored at 4℃ for 6 months. [Conclusion] In this study, the established gold immunochromatographic strip test kit is simple, rapid, sensitive and specific for detecting FMDV O, A, Asia I type, and is potentially useful for the first time and on-the-spot diagnosis.