Viral Disease Division, Animal, Plant and Fisheries Quarantine and Inspection Agency, 175 Anyang-ro, Manan-gu, Anyang-si, Gyeonggi-do 430-757, Republic of Korea.
J Virol Methods. 2011 Dec;178(1-2):31-8. doi: 10.1016/j.jviromet.2011.08.008. Epub 2011 Aug 19.
A microfluidic immunosensor utilizing Mie scattering immunoaggultination assay was developed for rapid and sensitive detection of porcine reproductive and respiratory syndrome virus (PRRSV) from lung tissue samples of domesticated pigs. Antibodies against PRRSV were conjugated to the surface of highly carboxylated polystyrene microparticles (diameter=920nm) and mixed with the diluted PRRSV tissue samples in a Y-shaped microchannel. Antibody-antigen binding induced microparticle immunoagglutination, which was detected by measuring the forward 45° light scattering of 380nm incident beam using microcallipered, proximity fiber optics. For comparison, multi-well experiments were also performed using the same optical detection setup. The detection limit was determined to be 10(-3)TCID(50)ml(-1) for PRRSV dissolved in PBS, while those of previous RT-PCR studies for PRRSV were 10(1)TCID(50)ml(-1) (conventional assays) or <1TCID(50)ml(-1) (quantitative real-time assays). Mie scattering simulations were able to predict the shape of the PRRSV standard curve, indicating that any non-linearity of the standard curve can be interpreted purely as an optical phenomenon. Each assay took less than 5min. A strong correlation could be found between RT-PCR and this method for the lung tissue samples, even though their respective detection mechanisms are different fundamentally (nucleic acids for RT-PCR and virus antigens for light scattering immunoagglutination assay). Several different dilution factors were also tested for tissue samples, and 1/10 and 1/100 were found to be usable. If the microfluidic chips are used only once (i.e. without re-using them), both superior sensitivity and satisfactory specificity can be demonstrated. Specificity studies revealed the presence of Type II PRRSV and non-presence of Type I PRRSV and that the microfluidic chip assay could detect Type II North American strain of PRRSV for the animals tested. This work demonstrates the potential of the Mie scattering immunoassay on a microfluidic chip towards real-time detection system for viral pathogens in domesticated animals.
一种基于米氏散射免疫凝集分析的微流控免疫传感器被开发出来，用于快速、灵敏地检测来自家养猪肺部组织样本中的猪繁殖与呼吸综合征病毒（PRRSV）。针对 PRRSV 的抗体被连接到高度羧化的聚苯乙烯微球（直径=920nm）的表面，并与稀释的 PRRSV 组织样本在 Y 型微通道中混合。抗体-抗原结合诱导微球免疫凝集，通过使用微卡尺近纤维光学测量 380nm 入射光束的前向 45°光散射来检测。为了进行比较，还使用相同的光学检测装置进行了多孔实验。对于溶解在 PBS 中的 PRRSV，检测限确定为 10(-3)TCID(50)ml(-1)，而之前针对 PRRSV 的 RT-PCR 研究的检测限为 10(1)TCID(50)ml(-1)（常规检测）或 <1TCID(50)ml(-1)（定量实时检测）。米氏散射模拟能够预测 PRRSV 标准曲线的形状，表明标准曲线的任何非线性都可以纯粹解释为光学现象。每次检测都不到 5 分钟。即使它们的检测机制在根本上不同（RT-PCR 用于核酸，而光散射免疫凝集分析用于病毒抗原），对于肺部组织样本，也可以发现 RT-PCR 与该方法之间存在很强的相关性。还测试了针对组织样本的不同稀释因子，发现 1/10 和 1/100 可用。如果微流控芯片仅使用一次（即不再重复使用），则可以同时证明更高的灵敏度和令人满意的特异性。特异性研究表明存在 II 型 PRRSV 而不存在 I 型 PRRSV，并且微流控芯片检测可用于测试动物的 II 型北美 PRRSV 毒株。这项工作展示了微流控芯片上的米氏散射免疫分析在用于检测家养动物中病毒病原体的实时检测系统方面的潜力。
