一种改良的布鲁氏菌病抗体微量凝集检测方法的建立与评价
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摘要
目的建立一种微量的布鲁氏菌病抗体凝集检测方法,用于布鲁氏菌病高通量检测。方法依据我国《布鲁氏菌病诊断标准》(WS269-2007)规定的病人诊断标准,用试管凝集试验做诊断标准。通过优化微量凝集试验抗原浓度,对142份疑似病人血清同时做试管凝集和微量凝集试验,进行效果评价。结果最优的微量凝集抗原浓度为1∶10,建立微量凝集法具有较高的敏感度和特异度,分别为98.9%和92.3%;和常规试管凝集法相比,两种方法符合率为96.5%。常规试管凝集检测得到21份阴性样本,22份可疑样本(1∶50),99份阳性样本(>1∶100)。微量凝集检测得到30份阴性样本、17份可疑样本(1∶50)、95份阳性样本(>1∶100)。两种试验方法,结果相同的占70.4%(100/142),经统计学检验差异无统计学意义(χ~2=0.8,P>0.05)。结论建立一种可显色的布鲁氏菌病抗体微量凝集检测方法,可以在96孔V型板上同时检测24份标本,且结果易判读,更适宜基层防疫人员现场检测,最终为疫情处置提供强有力的技术支持。
Although the serum tube agglutination test(SAT) is the standardized gold method, it is laborious, time consuming, and requires a number of reagents. A modified microagglutination test(MAT) variant of the SAT is evaluated for serological diagnoses. Ninety SAT-positive sera and 52 SAT-negative sera were used in this study. Antibody titers of 1∶100(++) were considered positive readings in both the SAT and MAT. Brucella abortus 104 antigens and Brucella-positive control antiserum were used in the SAT and MAT. The titers of the MAT differed according to antigen concentration.The optimal concentration of B.abortus 104 antigen(1∶10) was determined to compare the sensitivity and specificity between the MAT and SAT.The sensitivity and specificity of the MAT were 98.9% and 92.3% with reference to SAT. The MAT is less time consuming and requires less antigen and serum than the SAT. Results of the MAT showed good agreement with those of SAT by χ~2 test. The results of this study suggest that the MAT could be useful for diagnosis of brucellosis. For the simple and rapid diagnosis of brucellosis, the MAT is standardized using samples for the SAT to define positive and negative categories, and compare the sensitivity and specificity of the MAT and SAT.
Although the serum tube agglutination test(SAT) is the standardized gold method, it is laborious, time consuming, and requires a number of reagents. A modified microagglutination test(MAT) variant of the SAT is evaluated for serological diagnoses. Ninety SAT-positive sera and 52 SAT-negative sera were used in this study. Antibody titers of 1∶100(++) were considered positive readings in both the SAT and MAT. Brucella abortus 104 antigens and Brucella-positive control antiserum were used in the SAT and MAT. The titers of the MAT differed according to antigen concentration.The optimal concentration of B.abortus 104 antigen(1∶10) was determined to compare the sensitivity and specificity between the MAT and SAT.The sensitivity and specificity of the MAT were 98.9% and 92.3% with reference to SAT. The MAT is less time consuming and requires less antigen and serum than the SAT. Results of the MAT showed good agreement with those of SAT by χ~2 test. The results of this study suggest that the MAT could be useful for diagnosis of brucellosis. For the simple and rapid diagnosis of brucellosis, the MAT is standardized using samples for the SAT to define positive and negative categories, and compare the sensitivity and specificity of the MAT and SAT.
引文
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[4] 王淑云,刘熹,荣蓉,等.五种布鲁菌血清学检测方法对比分析[J]. 中华预防医学杂志, 2016,(2):175-178.
[5] Park, SH, Lee YH, Chu H et al.Application of the microagglutination test for serologic diagnosis of human brucellosis[J]. Osong Public Health Res Perspect, 2012, 3(1):19-23. DOI: 10.1016/j.phrp.2012.01.003
[6] Gomez, MC, Nieto JA, Rosa C et al.Evaluation of seven tests for diagnosis of human brucellosis in an area where the disease is endemic[J]. Clin Vaccine Immunol, 2008, 15(6):1031-1033. DOI: 10.1128/CVI.00424-07
[7] Galinska EM, Zagorski J. Brucellosis in humans--etiology, diagnostics, clinical forms[J]. Ann Agric Environ Med,2013,20(2):233-238.
[8] Konrad JL, Campero LM, Caspe GS, et al. Detection of antibodies against Brucella abortus, Leptospira spp. and Apicomplexa protozoa in water buffaloes in the Northeast of Argentina[J]. Trop Anim Health Prod,2013,45(8):1751-1756. DOI: 10.1007/s11250-013-0427-y
[9] Baum, M, Zamir O, Bergman-Rios R et al.Comparative evaluation of microagglutination test and serum agglutination test as supplementary diagnostic methods for brucellosis[J]. J Clin Microbiol, 1995, 33(8):2166-2170.
[10] Sayan M, Erdenlig S, Stack J, et al. A serological diagnostic survey for Brucellacanis infection in Turkish patients with Brucellosis-like symptoms[J].Jpn J Infect Dis,2011,64(6):516-519.
[11] de Oliveira, MZ, Vale V, Keid L,et al.Validation of an ELISA method for the serological diagnosis of canine brucellosis due to Brucella canis[J]. Res Vet Sci, 2011, 90(3):425-431. DOI: 10.1016/j.rvsc.2010.07.004
[12] 夏邦世, 吴金华.Kappa一致性检验在检验医学研究中的应用[J]. 中华检验医学杂志, 2006,29(1):83-84.
[2] 尚德秋.布鲁氏菌病再度肆虐及其原因[J]. 中国地方病防治杂志, 2001,16(01):29-34.
[3] 栾旭波.布鲁氏菌病血清学诊断方法及评价[J]. 中国畜牧兽医文摘, 2017,(8):63.
[4] 王淑云,刘熹,荣蓉,等.五种布鲁菌血清学检测方法对比分析[J]. 中华预防医学杂志, 2016,(2):175-178.
[5] Park, SH, Lee YH, Chu H et al.Application of the microagglutination test for serologic diagnosis of human brucellosis[J]. Osong Public Health Res Perspect, 2012, 3(1):19-23. DOI: 10.1016/j.phrp.2012.01.003
[6] Gomez, MC, Nieto JA, Rosa C et al.Evaluation of seven tests for diagnosis of human brucellosis in an area where the disease is endemic[J]. Clin Vaccine Immunol, 2008, 15(6):1031-1033. DOI: 10.1128/CVI.00424-07
[7] Galinska EM, Zagorski J. Brucellosis in humans--etiology, diagnostics, clinical forms[J]. Ann Agric Environ Med,2013,20(2):233-238.
[8] Konrad JL, Campero LM, Caspe GS, et al. Detection of antibodies against Brucella abortus, Leptospira spp. and Apicomplexa protozoa in water buffaloes in the Northeast of Argentina[J]. Trop Anim Health Prod,2013,45(8):1751-1756. DOI: 10.1007/s11250-013-0427-y
[9] Baum, M, Zamir O, Bergman-Rios R et al.Comparative evaluation of microagglutination test and serum agglutination test as supplementary diagnostic methods for brucellosis[J]. J Clin Microbiol, 1995, 33(8):2166-2170.
[10] Sayan M, Erdenlig S, Stack J, et al. A serological diagnostic survey for Brucellacanis infection in Turkish patients with Brucellosis-like symptoms[J].Jpn J Infect Dis,2011,64(6):516-519.
[11] de Oliveira, MZ, Vale V, Keid L,et al.Validation of an ELISA method for the serological diagnosis of canine brucellosis due to Brucella canis[J]. Res Vet Sci, 2011, 90(3):425-431. DOI: 10.1016/j.rvsc.2010.07.004
[12] 夏邦世, 吴金华.Kappa一致性检验在检验医学研究中的应用[J]. 中华检验医学杂志, 2006,29(1):83-84.
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