A Reagent for Rapid and Effective Virus Isolation and Concentration

描述（由申请人提供）：目前全球应对艾滋病毒/艾滋病流行病的努力侧重于预防新的感染和护理艾滋病毒感染者。大大改善了艾滋病毒/艾滋病患者前景的高效抗逆转录病毒疗法（HAART）费用昂贵，但正通过慈善组织和制药公司逐步向发展中国家提供这种疗法。然而，为了使其有效，必须对HAART进行监测，以避免快速发展和传播耐药菌株的风险。监测HIV病毒载量或血浆中病毒RNA的浓度是HIV感染者标准护理的一部分。HIV/AIDS患者护理中的两个关键决定主要基于HIV病毒载量测量：何时开始药物治疗以及何时转换或调整药物治疗方案。类似地，HBV和HCV病毒载量也被用于监测疾病进展和治疗效果。核酸检测技术的改进，如实时PCR的引入，大大简化了病毒载量检测。然而，从血清或血浆样品制备核酸仍然是一个困难和昂贵的过程，这在一定程度上阻碍了这些技术在发展中国家的采用。我们现在已经开发了一种病毒捕获试剂-涂有化学聚合物的磁性颗粒，其似乎对于从甚至高度稀释的人血清样品中分离和浓缩HIV-1、HBV和HCV非常有效，例如，20个RNA拷贝或25 IU/mL。分离的病毒可以在PCR相容的缓冲液中裂解，并直接用于使用PCR或其他技术的检测。拟议的研究旨在严格评估该试剂，并制定一个简化的样品制备方案，更便宜，更有效，适合资源贫乏地区。如果成功开发，整个样品制备将需要不到30分钟，仅使用磁铁就需要5-10分钟的劳动。使用这种样品制备方法，应该可以在不到15分钟的时间内进行实时PCR病毒载量测试，而无需使用昂贵的自动化样品制备系统。拟议的研究将评估使用聚合物涂层磁性颗粒从人血清或血浆样本中分离和浓缩病毒（HIV-1，HBV和HCV）的可行性，作为一种简化的样本制备方法，更便宜，更有效，适用于资源贫乏地区。临床和人工样本（标准品）将用于确定试剂在检测病毒载量时的灵敏度和一致性。

DESCRIPTION (provided by applicant): Current global efforts to confront the HIV/AIDS epidemic focus on the prevention of new infections and the care for those who are living with HIV. Highly active antiretroviral therapy (HAART) which has dramatically improved the outlook of HIV/AIDS patients is expensive although it is gradually being made available to developing countries through charitable organizations and pharmaceutical companies. For it to be effective, however, HAART has to be monitored to avoid the risk of rapid development and transmission of resistant strains. Monitoring HIV viral load, or the concentration of viral RNA in the plasma, is part of the standard care of HIV-infected individuals. Two critical decisions in HIV/AIDS patient care are primarily based on HIV viral load measurements: when to initiate drug therapy and when to switch or adjust drug therapy regimen. Similarly, HBV and HCV viral loads have also been used to monitor disease progression and treatment efficacy. Improvements of nucleic acid detection technologies such as the introduction of real-time PCR have considerably simplified viral load testing. However, preparation of nucleic acids from serum or plasma samples remains a difficult and expensive process that, in part, hinders the adoption of these technologies in developing countries. We have now developed a virus capture reagent - magnetic particles coated with a chemical polymer, which seems to be highly effective for isolating and concentrating HIV-1, HBV and HCV from even highly diluted human serum samples, e.g., 20 RNA copies or 25 IU/mL. Isolated viruses could be lysed in a PCR compatible buffer and directly used for detection using PCR or other technologies. The proposed studies are aimed to critically evaluate this reagent and to develop a protocol for simplified sample preparation that is less expensive, more effective and suitable for resource-poor regions. If successfully developed, the entire sample preparation will take less than 30 minutes with 5-10 minute labor using only a magnet. With this sample preparation method, it should be possible to perform a real-time PCR viral load test with less than 15 minutes labor and without using an expensive automated sample preparation system. The proposed studies will evaluate the feasibility of using polymer coated magnetic particles to isolate and concentrate viruses (HIV-1, HBV and HCV) from human serum or plasma samples as a simplified sample preparation method that is less expensive, more effective and suitable for resource-poor regions. Clinical and contrived samples (standards) will be used to establish sensitivity and consistency of the reagent in detecting the viral load.