• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

鸭短喙矮小综合征病毒抗体间接ELISA检测方法的建立与初步应用

An Indirect ELISA for Diagnosing Duck Short Beak and Dwarfism Syndrome

  • 摘要:
    目的 建立一种快速检测鸭短喙矮小综合征病毒(Short beak and dwarfism syndrome virus, SBDSV)抗体的血清学方法。
    方法 以蔗糖密度梯度离心纯化的SBDSV流行株 M15,以此灭活全病毒作为固相包被抗原,通过系统性优化反应参数,构建一种高特异性的间接 ELISA 检测方法,用于鸭短喙矮小综合征病毒(SBDSV)血清抗体的定性分析。
    结果 建立的间接ELISA检测方法的最佳反应条件为:抗原包被浓度0.1 mg·mL−1,37 ℃孵育2 h后再4 ℃过夜包被;1% BSA 37 ℃封闭2 h;血清样本1∶200稀释;阴阳性临界值为0.3214。该检测方法特异性好,与其他常见水禽病毒阳性血清均无交叉反应;敏感性高,阳性高免血清(LPAI效价28)1∶ 204 800倍稀释时检测结果仍为阳性;批内、批间重复性好,变异系数均小于6%;利用所建立的ELISA方法对70份SBDSV疑似感染病鸭的血清样品进行检测,检测结果与LPAI检测结果符合率为90%。对福建省648份临床送检1日龄雏鸭血清样品进行检测,抗体阳性率约为48.30%,显示福建省养鸭场存在不同程度的SBDSV感染。
    结论 本研究建立的SBDSV血清抗体间接ELISA检测方法,可为监测SBDSV在我国的流行情况以及雏鸭母源抗体水平评价提供技术支撑。

     

    Abstract:
    Objective A rapid serological method was developed for detecting antibodies against the Short Beak and Dwarf Syndrome Virus (SBDSV) in ducks.
    Methods The epidemic strain M15 of SBDSV was purified by sucrose density gradient centrifugation and inactivated to be used as the solid-phase coating antigen. After optimization of reaction conditions, a highly specific indirect enzyme-linked immunosorbent assay (ELISA) was developed and verified by testing for assay specificity, repeatability, and accuracy on clinic specimens for detecting the specific antibody in ducks.
    Results The optimal ELISA analytical conditions included the uses of antigen at the concentration of 0.1 mg·mL−1, coating the antigen at 37 ℃ for 2 h followed by incubation overnight at 4 ℃ and blocked with 1% BSA at 37 ℃ for 2 h, serum dilution at the rate of 1∶200, and 0.3214 as the positive/negative cutoff point. The newly developed assay exhibited high specificity with no cross-reactivity on other common waterfowl viruses. It was highly sensitive as shown by remaining positive on hyperimmune serum (with an LPAI titer of 28) up to 1∶204 800 dilution. The repeatability on the intra- and inter-assays was high with coefficients of variation below 6%. On 70 serum specimens of ducks suspected of SBDSV infection, the assay delivered 90% of the results agreed with those obtained by LPAI. On the 648 serum samples of 1-day-old ducklings submitted for testing in Fujian, an antibody positivity rate of approximately 48.30% was detected by the new assay indicating some degrees of SBDSV infection on the birds at duck farms in the province.
    Conclusion The indirect ELISA for detecting SBDSV serum antibodies newly developed in this study could facilitate a rapid and effective monitoring means on epidemic SBDSV in the country and, in general, provide a technic support for the detection of the viral infection in ducklings.

     

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