布鲁氏菌病荧光偏振抗体检测方法的建立

Development of fluorescence polarization method in Brucellosis antibody detection

摘要:

【目的】布鲁氏菌病(Brucellosis)简称布病，是由布鲁氏菌引起的以感染家畜为主的人畜共患传染病，造成严重的公共卫生问题。目前全世界范围内消除该病的主要方法是扑杀与免疫相结合，所以建立快速准确的诊断方法对防治和清除布病非常必要。本文建立布鲁氏菌病荧光偏振(FPA)抗体检测方法，为布鲁氏菌病(布病)的快速高效诊断提供技术手段。【方法】提纯猪种布鲁氏菌S2株脂多糖O链(OPS)，经异硫氰酸荧光素(FITC)标记后作为诊断抗原。通过对样品稀释液、抗原稀释度、反应时间等条件的优化，初步建立了布鲁氏菌荧光偏振诊断方法。用该方法对148份布病阳性血清(其中牛血清70份，羊血清78份)和155份布病阴性血清(其中牛血清82份，羊血清73份)进行检测，确定其敏感性和特异性。按确定的技术参数，制备3批布鲁氏菌FPA抗体检测试剂盒，使用质控阴、阳性血清分别评价试剂盒的批内和批间重复性。用400份临床样本比较本研究开发试剂盒与商品化进口FPA试剂盒的符合率。【结果】使用0.5%蔗糖磷酸缓冲液作为血清样品稀释液；标记抗原的使用浓度为90 μg/ml；最佳反应时间为3?5 min。本检测方法的判定标准为：δmP值<20时为阴性，δmP值≥20时为阳性。按上述条件建立的FPA检测148份布病阳性血清和155份布病阴性血清，结果敏感性为98.6%，特异性为98.7%。对400份临床样本的比对检测显示，研究建立的FPA方法与进口商品化试剂盒的总符合率为94.0%。【结论】研究建立的布鲁氏菌PFA抗体检测方法具有良好的特异性和敏感性，可作为一种重要的布病诊断快速诊断方法。

Abstract:

[Objective] Brucellosis (Brucellosis) referred to as cloth disease, is caused by Brucella to livestock-based zoonotic infectious diseases, causing serious public health problems. At present, the main method of eliminating the disease around the world is the combination of culling and immunization, so the establishment of rapid and accurate diagnostic methods for the prevention and removal of brucellosis is necessary. We establish a diagnostic method of fluorescence polarization (FPA) for brucellosis and provide a scientific and efficient method for diagnosis of brucellosis. [Methods] In the present study, the conjugate of lipopolysaccharide O-chain (OPS) and fluorescein isothiocyanate (FITC) was purified from purified S2 strain of Brucella spp. As an antigen, and the optimal dilution, dilution concentration, reaction conditions, the results to determine parameters such as the initial establishment of the Brucella fluorescence polarization diagnosis method. The results showed that the sensitivity and specificity of the positive serum of 148 bovine serum (including 70 bovine serum and 78 goat serum) and 155 negative bovine serum (including 82 bovine serum and 73 goat serum) were determined by this method. The intra-assay and inter-assay reproducibility was assessed using the controlled positive and negative serum assay kits. At the same time, 400 samples of bovine serum samples were detected by FPA kit and commercial kit, and the coincidence rate was compared. [Results] The optimal conditions of each component in the kit were: the best sample dilution was 0.5% sucrose phosphate buffer; the concentration of the labeled antigen was 90 μg/mL; the best reaction time was 3 to 5 minutes; δmP value of <20, was negative, δmP value of ≥20, was positive. The sensitivity of the method was 98.6% and the specificity was 98.7%. The comparison of 400 clinical samples showed that the FPA method established in this study coincided with the import commercialization kit 94.0%. [Conclusion] The method of fluorescence polarization diagnosis with good specificity and sensitivity was established.