量子点标记免疫层析技术检测布鲁氏菌病的方法研究
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摘要
目的通过研究量子点标记免疫层析技术检测布鲁氏菌病抗体,制备量子点免疫层析试纸条,验证快速检测布鲁氏菌病的可行性。方法将蛋白A和布鲁氏菌全菌蛋白分别作为质控线和检测线喷涂在硝酸纤维素膜上,以量子点标记布鲁氏菌全菌蛋白,稀释后喷涂在玻璃纤维素膜上,最后进行组装、切割制成检测用试纸条。应用该试纸条检测布鲁氏菌病患者样本102例,健康人血清47例,以试管凝集试验为对照,比较两组方法的符合率。结果建立的量子点免疫层析试纸条性能较好,布鲁氏菌病抗体的检测敏感性为99.0%,特异度为95.8%,两者符合率为98.0%;将阳性血清稀释至128倍,通过荧光检测仪仍可检测到T线荧光值,该纸条重复性好,储存时间30 d内稳定性较好,其变异系数为4.1%。结论该方法操作简单、快速稳定、灵敏度高、成本低,15 min内完成检测,血清用量低,可实现现场快速检测,在布鲁氏菌病的早期检测中具有良好前景。
In this study, we prepared quantum dots immunochromatographic test strips for the detection of brucellosis antibodies by immunochromatography and studied the feasibility of rapid detection of brucellosis. Protein A and Brucella Whole Protein were sprayed on nitrocellulose membranes as control lines and detection lines respectively. Brucella Whole Protein were labeled with quantum dots diluted and sprayed on glass cellulose membranes. Paste the sample pad, release pad, reaction membrane and absorbent pad on the backing card in the corresponding order and cut them into test strips. We collected 149 effective serum samples,including 102 clinical samples of brucellosis patients and 47 healthy human sera. The Tube Agglutination Test(SAT)was used as control and the coincidence rates of the two methods were compared. The results showed that the established quantum dots immunochromatographic strip has good performance. The detection sensitivity of brucellosis antibody is 99.0%, the specificity is 95.8%, and the coincidence rate is 98.0%. The positive serum was diluted to 128 times, and T-line fluorescence value was detected by fluorescence detector. The strips prepared in the same batch were tested for brucellosis positive serum within 30 days, and the coefficient of variation was 4.1%. It is indicated that this method is simple, rapid, stable, sensitive, and low cost. Under optimized conditions, the concentration of brucellosis antibodies could be determined within 15 min with high sensitivity and specificity using only 10 μL of sample. Therefore, it can realize rapid on-site detection and has a good prospect in the early detection of brucellosis.
In this study, we prepared quantum dots immunochromatographic test strips for the detection of brucellosis antibodies by immunochromatography and studied the feasibility of rapid detection of brucellosis. Protein A and Brucella Whole Protein were sprayed on nitrocellulose membranes as control lines and detection lines respectively. Brucella Whole Protein were labeled with quantum dots diluted and sprayed on glass cellulose membranes. Paste the sample pad, release pad, reaction membrane and absorbent pad on the backing card in the corresponding order and cut them into test strips. We collected 149 effective serum samples,including 102 clinical samples of brucellosis patients and 47 healthy human sera. The Tube Agglutination Test(SAT)was used as control and the coincidence rates of the two methods were compared. The results showed that the established quantum dots immunochromatographic strip has good performance. The detection sensitivity of brucellosis antibody is 99.0%, the specificity is 95.8%, and the coincidence rate is 98.0%. The positive serum was diluted to 128 times, and T-line fluorescence value was detected by fluorescence detector. The strips prepared in the same batch were tested for brucellosis positive serum within 30 days, and the coefficient of variation was 4.1%. It is indicated that this method is simple, rapid, stable, sensitive, and low cost. Under optimized conditions, the concentration of brucellosis antibodies could be determined within 15 min with high sensitivity and specificity using only 10 μL of sample. Therefore, it can realize rapid on-site detection and has a good prospect in the early detection of brucellosis.
引文
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[12] 朱明东,杨蓉,洪林娣,等.快速诊断布鲁氏菌病胶体金免疫层析法的建立[J].中国卫生检验杂志,2008,18(7):1344-1355.
[13] 陈珍珍,赵越,王占黎,等.自制胶体金试剂盒对布鲁氏菌病的诊断效果评价[J].现代预防医学,2016,43(15):2802-2805.
[14] 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,2016,50(2):175-178.DOI:10.3760/cma.j.issn.0253-9624.2016.02.014
[2] Matyas Z,Fujikura T.Brucellosis as a world problem[J].Dev Biol Stand,1984,56(7):3-20.
[3] Kaden R,Ferrari S,Alm E,et al.A novel real-time PCR assay for specific detection of Brucella melitensis[J].BMC Infect Dis,2017,17(1):230-236.DOI:10.1186/s12879-017-2327-7
[4] Song DD,Qu XF,Liu YS,et al.A rapid detection method of Brucella with quantum dots and magnetic beads conjugated with different polyclonal antibodies[J].Nanoscale Res Lett,2017,12(1):179-188.DOI:10.1186/s11671-017-1941-z
[5] 王素梅,蒋治良,梁爱惠.CdTe量子点的光谱特性及其应用[J].光谱学与光谱分析,2008,28(9):2152-2155.
[6] Qiu L,Bi Y,Wang C,et al.Protein a detection based on quantum dots antibody bioprobe using fluorescence coupled capillary electrophoresis [J].Int J Mol Sci,2014,15(2):1804-1811.DOI:10.3390/ijms15021804
[7] Mulder WJ,Castermans K,van Beijnum JR,et al.Molecular imaging of tumor angiogenesis using alphavbeta3-integrin targeted mμLtimodal quantum dots[J].Angiogenesis,2009,12(1):17-24.DOI:10.1007/s10456-008-9124-2
[8] Wang L,Zhang JX,Bai HL,et al.Specific Detection of Vibrio Parahaemolyticus by Fluorescence Quenching Immunoassay Based on Quantum Dots[J].Appl Biochem Biotechnol,2014,173 (5):1073-1082.DOI:10.1007/s12010-014-0904-4
[9] Cheng X,Pu X,Jun P.Rapid and quantitative detection of C-reactive protein using quantum dots and immunochromatographic test strips [J].Int J Nanomed,2014,2014 (1):5619-5926.DOI:10.2147/IJN.S74751
[10] 林彦星,曹琛福,张彩虹,等.非洲猪瘟病毒抗体量子点检测试纸条的研制[J].中国兽医科学,2017,47(10):1214-1220.
[11] 黄培,韩秋雪,胡星星,等.中东呼吸综合征冠状病毒量子点免疫层析试纸的制备[J].中国病原生物学杂志,2018,13(6):557-561.
[12] 朱明东,杨蓉,洪林娣,等.快速诊断布鲁氏菌病胶体金免疫层析法的建立[J].中国卫生检验杂志,2008,18(7):1344-1355.
[13] 陈珍珍,赵越,王占黎,等.自制胶体金试剂盒对布鲁氏菌病的诊断效果评价[J].现代预防医学,2016,43(15):2802-2805.
[14] 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,2016,50(2):175-178.DOI:10.3760/cma.j.issn.0253-9624.2016.02.014