布鲁氏菌病微量补体结合试验的方法改进和应用

Method Improvement and Its Application of Micro Complement Fixation Test for Brucellosis

【目的】补体结合试验是布鲁氏菌病(简称“布病”)血清学确诊方法。现行国家标准《动物布鲁氏菌病诊断技术》(GB/T 18646-2018)中收录的补体结合试验分为常量法补体结合试验(CFT)和微量法补体结合试验(mCFT),但两者之间的相关技术参数和结果判定标准均存在差异,导致常量法CFT和mCFT检测结果不一致。笔者通过对mCFT参数的改进优化,使现行标准中mCFT的检测结果等效于常量法CFT,实现布病CFT的高通量准确诊断,对现有布病检测方法进行了补充。【方法】参照布病常量法CFT反应条件和诊断标准,对现行国标中mCFT的反应时间、稀释液、效价测定方法、结果判定等相关参数进行改进优化。改进后的mCFT方法与原mCFT方法相比,效价测定中受检血清的稀释度由1﹕2-1﹕64稀释改为1﹕10稀释,稀释方法与常量法CFT相同,确保微量法的检测临界值与常量法一致;采用酶标仪读取OD600值判定溶血强度,降低了试验人员肉眼判定的误差,提升了结果判定的效率和准确性;反应时间由37℃水浴30 min缩短为20 min,提高了检测效率又不影响检测结果;稀释液由巴比妥缓冲液改为生理盐水,与常量法使用相同稀释液并可获得理想结果。用改进的mCFT与国标的mCFT、常量法CFT分别对来自内蒙古、山东、北京、江苏等地区的409份临床牛、羊血清样品(牛血清163份、羊血清246份)进行检测,分析比较其符合率。【结果】采用改进的mCFT与常量法CFT对409份临床样品进行比对检测,改进的mCFT方法检出53份阳性、11份可疑、345份阴性;常量法CFT检出53份阳性、9份可疑、347份阴性。比较两者检测结果,只有2份牛血清样品用改进的mCFT方法检测为可疑,而常量法CFT检测为阴性,其余407份样品检测结果均一致。其中牛血清样品的符合率98.77%,羊血清样品的符合率100%,总符合率99.51%。而国标mCFT方法因阳性判定标准低于常量法CFT,且不设可疑区间,结果检出97份阳性、312份阴性。与常量法CFT结果相比较,牛血清样品的符合率88.34%,羊血清样品的符合率89.84%,总符合率为89.24%,远低于本研究改进的mCFT方法与常量法CFT 99.51%的符合率。【结论】通过对mCFT方法各参数的优化,建立了一种与常量法CFT判定标准一致,且检测结果高度符合的mCFT方法。

【Objective】The complement fixation test is used as a confirmatory test for the serodiagnosis of brucellosis.The complement fixation tests are divided into constant complement fixation test(CFT)and micro complement fixation test(mCFT)in diagnostic techniques for animal brucellosis(GB/T 18646-2018).However,there are differences in the related technical parameters and judgment of the result between them,which lead to the inconsistency of CFT and mCFT results.In this study,By the improvement and optimization of the parameters of m CFT,this study made the detection results of m CFT equivalent to classical CFT,and achieved the high-throughput and accurate diagnosis of CFT for brucellosis.【Method】According to classical CFT reaction conditions and diagnostic criteria,the reaction time,diluent,titer determination,result judgment and other related parameters for the current national standard of m CFT were optimized and improved.Compared with the original method,the dilution of the tested serum was changed from 1:2-1:64 to 1:10.The dilution method was the same as classical CFT so that the detection critical value was consistent with the constant method.The OD600 value was read by the microplate reader to determine the hemolysis intensity,which reduced the error caused by visual judgment and improved the efficiency and accuracy of result judgment.The reaction time was shortened from 30 min to 20 min in water bath at 37℃,which improved the detection efficiency without affecting the results.The diluent was changed from barbital buffer to normal saline,which used the same diluent of classical CFT and achieved satisfactory results.The 409 clinical bovine and sheep serum samples(163 bovine serum samples and 246 sheep serum samples)from Inner Mongolia,Shandong,Beijing and Jiangsu were detected by improved mCFT,the national standard mCFT,and classical CFT,respectively.The coincidence rate was analyzed and compared.【Result】By detecting the 409 clinical samples,the result showed that 53 samples were positive,11 samples were suspicious and 345 were negative by improved mCFT.The 53 positive samples,9 suspicious samples and 347 negative samples were detected by classical CFT.Compared with the results of the two methods,only two bovine serum samples were suspicious by the improved mCFT,which were negative by classical CFT.The results of the other 407 samples were consistent.The coincidence rate of bovine serum samples was 98.77%,which of sheep serum samples was 100%,and the total coincidence rate was 99.51%between improved mCFT and classical CFT.However,the positive criteria of national standard m CFT were lower than that of classical CFT,and no suspicious interval was set.The results showed that 97samples were positive and 312 samples were negative.Compared with the results of classical CFT,the coincidence rate of bovine serum samples was 88.34%,which of sheep serum samples was 89.84%;the total coincidence rate was 89.24%,which was far lower than 99.51%of improved mCFT and classical CFT.【Conclusion】By optimizing the parameters of mCFT,the mCFT method was established,which was highly consistent with the criterion and the results of the classical CFT.