布鲁氏菌病微量补体结合试验的方法改进和应用

doi:10.3864/j.issn.0578-1752.2022.08.017

Abstract

Abstract:

【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 mCFT, this study made the detection results of mCFT 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 mCFT 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 mCFT were lower than that of classical CFT, and no suspicious interval was set. The results showed that 97 samples 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.

Key words: brucellosis, micro complement fixation test, method improvement, application

JIANG Hui,FENG Yu,QIN YuMing,ZHU LiangQuan,FAN XueZheng,DING JiaBo. Method Improvement and Its Application of Micro Complement Fixation Test for Brucellosis[J].Scientia Agricultura Sinica, 2022, 55(8): 1676-1684.

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EP管号 EP tube No.	1	2	3	4	5	6	7	8	9	10
1﹕500稀释溶血素 1﹕500 hemolysin	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
生理盐水Saline	0.1	0.2	0.3	0.4	0.5	0.6	0.7	0.8	0.9	1.0
稀释度Dilution	1﹕1000	1﹕1500	1﹕2000	1﹕2500	1﹕3000	1﹕3500	1﹕4000	1﹕4500	1﹕5000	1﹕5500

检测方法 Detection method	血清种类 Serum type	总数 Total number	阳性数（阳性率） Positive number (Positive rate)	可疑数（可疑率） Suspicious number (Suspicious rate)	阴性数（阴性率） Negative number (Negative rate)	与常量法CFT符合率 Coincidence rate with the classical CFT
常量法CFT Classical CFT	牛血清Bovine serum	163	30 (18.41%)	7 (4.29%)	126 (77.3%)	/
	羊血清Sheep serum	246	23 (9.35%)	2 (0.81%)	221 (89.84%)	/
	合计Total	409	53 (12.96%)	9 (2.2%)	347 (84.84%)	/
国标mCFT National standard mCFT	牛血清Bovine serum	163	49 (30.06%)	0	114 (69.94%)	88.34%
	羊血清Sheep serum	246	48 (19.51%)	0	198 (80.49%)	89.84%
	合计Total	409	97 (23.72%)	0	312 (76.28%)	89.24%
改进mCFT Improved mCFT	牛血清Bovine serum	163	30 (18.41%)	9 (5.52%)	124 (76.07%)	98.77%
	羊血清Sheep serum	246	23 (9.35%)	2 (0.81%)	221 (89.84%)	100%
	合计Total	409	53 (12.96%)	11 (2.69%)	345 (84.35%)	99.51%

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

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