人及禽类禽流感病毒蛋白芯片检测方法建立  

Detection of avian influenza virus in birds and human using protein chip

作　　者：顾大勇[1] 徐云庆[1] 史蕾[1] 赵纯中[1] 刘春晓[1] 杨燕秋[1] 李永进[2] 林连成 

机构地区：[1]深圳出入境检验检疫局国际旅行卫生保健中心疾病预防控制研究室,广东深圳518033 [2]长江大学生命科学学院 [3]深圳市赛尔生物技术有限公司

出　　处：《中国公共卫生》2012年第1期71-73,共3页Chinese Journal of Public Health

摘　　要：目的建立可同时检测人及禽类禽流感病毒的蛋白芯片方法。方法采用醛基化玻璃载体蛋白芯片和双位点模式,将禽流感病毒H1、H3、H5、H7、H9、N1、N2、NP、NS1等9种亚型抗原以最佳浓度,点样于玻璃载体表面,构建相应抗体的检测芯片,分别用于禽类不同类型禽流感病毒血清及随机选择的人血清检测,并对特异性、敏感性及重复性进行测试,与血凝抑制法进行双向验证比较。结果探针H1、H3、H5、N1、N2、NP、NS1亚型抗原的最佳浓度为1 mg/mL,H7、H9亚型抗原最佳浓度为0.5 mg/mL;检测禽类禽流感病毒及人禽流感病毒使用的酶标抗体浓度分别为1∶1 000和1∶1 500;方法具有较好的特异性,对H7N7阳性血清的检测限为1∶40稀释度;重复性检测的变异系数均<2%;血凝抑制法和蛋白芯片法的检测结果一致。结论所建立的蛋白芯片法可用于人类和禽类禽流感病毒的检测。Objective To develop a protein chip-based method for simultaneous detection of avian influenza virus （AIV） from birds and human. Methods Protein chip was constructed by immobilizing the positive control（PC） ,negative control （NC） and different antigen subtypes with the optimal concentration onto the aldehyde glass slide according to the predesigned pattern. Then the prepared protein chip was used to detect the serum samples from the different AIV subtype and human. The parameters for the performance of protein chip ,such as specificity ,sensitivity and repeatability, were tested in details. In addition, interactive validation experiment was performed using the protein chip-based method and avian influenza virus （H subtype） hemagglutination inhibition test kit. Results To construct protein chip, the optimal probe concentrations of Hi, H3, H5, N1, N2 ,NP,NS1 and H7 ,H9 subtype were 1 mg/mL and 0.5 mg/mL,respectively. The optimal concentrations of horseradish peroxidase enzyme labeled antibody for AIV from birds and human were 1 ： 1000 and 1 ： 1500 dilution. The specificity of pre- pared protein chip was confirmed by testing the samples of H5N1, I-ITN7, H9N2 positive serum and normal serum. The results were consistent with the expected and there was no cross-reactivity observed. The prepared protein chip had a detec- tion limitation of 1：40 dilution for H7N7 positive serum and showed excellent repeatability validated by consecutive test of 10 times for H5, H7, and H9 positive serum with a variation coefficient of 〈 2%. The results of the interactive validation experiments using protein chip-based method were consistent with those of hemagglutination inhibition test. Conclusion The prepared protein chip has a good specificity, sensitivity, and repeatability and could be used to detect AIV from birds and human simultaneously.

关 键 词：蛋白芯片 禽流感 病毒 检测 

分 类 号：R373.1[医药卫生—病原生物学]