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【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),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90μg/mL;最佳反应时间为3-5 min。本检测方法的判定标准为:δm P值<20时,为阴性,δm P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。
【Purposes】 Brucellosis (Brucellosis) is a brucellosis-related zoonosis caused by brucellosis and causes serious public health problems. At present, the main method to eliminate the disease in the world is combination of culling and immunization. Therefore, it is necessary to establish a rapid and accurate diagnostic method for the prevention and treatment of brucellosis. In this paper, we established a method for the detection of brucellosis fluorescence polarization (FPA) antibody, which provides a technical means for the rapid and efficient diagnosis of brucellosis (brucellosis). 【Method】 The lipopolysaccharide O chain (OPS) of Brucella S2 strain was purified and labeled with fluorescein isothiocyanate (FITC) as diagnostic antigen. Through the optimization of the sample dilution, antigen dilution, reaction time and other conditions, the diagnostic method of Brucella fluorescence polarization was preliminarily established. In this method, 148 samples of brucellosis-positive sera (including 70 bovine serum, 78 sheep serum) and 155 brucella negative sera (including 82 bovine serum and 73 sheep serum) were tested to determine their sensitivity and specificity Sex. According to the determined technical parameters, three batches of Brucella anti-FPA antibody test kit were prepared, and the quality control positive and negative sera were used to evaluate the intra-assay and inter-assay reproducibility of the kit respectively. 400 clinical samples were used to compare the coincidence rate of this research and development kit with the imported FPA kit. 【Result】 0.5% sucrose phosphate buffer was used as a serum sample diluent. The concentration of labeled antigen was 90 μg / mL. The optimal reaction time was 3-5 min. The test method to determine the criteria: δm P value of <20, negative, δm P value of ≥ 20, was positive. According to the above conditions, FPA was used to detect 148 brucella positive sera and 155 brucellosis negative sera, with a sensitivity of 98.6% and a specificity of 98.7%. Alignment of 400 clinical samples showed that the overall coincidence rate of the established FPA method with the imported commercialization kit was 94.0%. 【Conclusion】 The detection method of Brucella PFA antibody has good specificity and sensitivity, which can be used as an important rapid diagnosis method of brucellosis diagnosis.