SIMULTANEOUS SCREENING FOR A/H3N2, A/H1N1, A/H5N1 AND B INFLUENZA VIRUSES

描述（由申请人提供）：我们的目标是开发一种商业上可行的流感诊断方法，用于快速、同时筛查临床样本中的甲型和B型流感以及重要亚型。与现有的流感病毒聚合酶链反应（PCR）方法相比，所提出的方法的优点源于使用单个基因片段来鉴定类型和亚型。所提出的工作的目标，基质基因片段，是已知的稳健扩增和更保守的比传统的血凝素基因（HA）的目标。开发产品的最初商业利基将是州和地方公共卫生实验室。虽然国家卫生实验室目前接受临床样本进行分型和亚型，但通过病毒分离和血凝抑制试验的“金标准”进行亚型分型的成本过高。目前，大多数卫生实验室依靠基于荧光的免疫测定来筛查H3或H1病毒，没有确认神经氨酸酶亚型，目前也没有新出现病毒的能力。那些利用实时逆转录PCR（RRT-PCR）检测的实验室必须依赖突变敏感的HA基因进行部分亚型分型，并且必须对每种HA亚型进行单独检测。拟议的产品将作为一种手段，以改善和扩大监测工作，在国家和地方一级在美国，以及在世界各地的区域实验室，通过提供一个快速和具有成本效益的手段，同时筛选型（A和B）和某些亚型，特别是目前的人类适应性流感病毒（A/H3 N2和A/H1 N1）和A/H5 N1。这种新的监测工具将用于取代现有的免疫测定和基于单重HA靶向RT-PCR的测定，但并不是为了取代病毒分离方法和测序，这是更全面了解流感病毒所必需的。 具体目标1将利用最近的发现，即流感病毒基因组的基质基因片段可以提供流感病毒的类型和亚型信息。待检验的假设是，可以设计一组用于RT-PCR的M基因片段特异性引物对（5），以选择性地检测A/H3 N2、A/H1 N1、A/H5 N1和B病毒。具体目标2将侧重于RT-PCR多重条件的系统优化。在Specific Aim 3中，满足初始样本成功标准的有希望的引物组将在一项盲法研究中进行验证，该研究采用多种采样方法（包括鼻拭子、鼻洗液和鼻咽抽吸）采集了至少两个流感季节的300多例患者样本。在第二阶段，我们将设计一个自动化样品处理系统，包括提取和RT-PCR扩增，然后通过快速色谱方法快速分离和检测PCR产物。预期PCR后分离和检测相对于多重RRT- PCR提供上级准确度和灵敏度，且不会增加整个检测的时间。 拟议工作的动机是流感病毒对人类和动物健康的巨大影响，以及对快速，廉价的菌株监测工具的需求。其目的是为州和地方公共卫生实验室提供使用高度可靠和保守的基质基因片段作为鉴定靶点来提供和快速筛查患者样本的流感类型和亚型的能力。

DESCRIPTION (provided by applicant): Our objective is to develop a commercially viable influenza diagnostic for rapid and simultaneous screening of clinical samples for influenza A and B type and important subtypes. The advantages of the proposed approach over existing polymerase chain reaction (PCR) methods for influenza viruses stem from the use of a single gene segment for identification of both type and subtype. The target for the proposed work, the matrix gene segment, is known to be robustly amplified and more conserved than the traditional hemagglutinin gene (HA) target. The initial commercial niche for the developed product will be state and local public health laboratories. While state health labs currently receive clinical samples for typing and subtyping, the cost of subtyping by the "gold standard" of viral isolation and hemagglutination inhibition test is prohibitive. Currently, most health labs rely on a fluorescence-based immunoassay to screen for H3 or H1 viruses, with no confirmation of the neuraminidase subtype and no current capabilities for emerging viruses. Those labs that utilize real-time reverse transcription PCR (RRT-PCR) assays must rely on the mutation- susceptible HA gene for partial subtyping and must conduct individual tests for each HA subtype. The proposed product would serve as a means to improve and broaden surveillance efforts at state and local levels in the US, as well as in regional labs worldwide, by providing a rapid and cost-effective means to simultaneously screen for type (A and B) and certain subtypes, specifically, current human-adapted influenza viruses (A/H3N2 and A/H1N1) and A/H5N1. This new surveillance tool would be used in place of existing immunoassays and singleplex HA targeted RT-PCR based assays but is not designed to replace viral isolation methods and sequencing, which are necessary for a more complete understanding of influenza viruses. Specific Aim 1 will capitalize on the recent discovery that the matrix gene segment of influenza's viral genome can provide both type and subtype information for influenza viruses. The hypothesis to be tested is that a small set of M gene segment specific primers pairs (5) for RT-PCR can be designed to selectively detect A/H3N2, A/H1N1, A/H5N1, and B viruses. Specific Aim 2 will focus on systematic optimization of multiplex conditions for RT-PCR. In Specific Aim 3, promising primer sets that satisfy the criteria for success with initial samples will be validated in a blind study of 300+ patient samples acquired over at least two flu seasons by a variety of sampling methods, including nasal swab, nasal wash, and nasopharyngeal aspiration. In Phase 2 we will engineer a system for automated sample handling, including extraction and RT-PCR amplification, followed by rapid separation and detection of PCR products by a fast chromatographic method. Post-PCR separation and detection is anticipated to provide superior accuracy and sensitivity relative to multiplex RRT- PCR with no added time to the overall assay. The motivation for the proposed work is the tremendous impact influenza viruses have on human and animal health and the need for rapid, inexpensive tools for strain surveillance. The intent is to provide state and local public health laboratories with the ability to affordably and rapidly screen patient samples for influenza type and subtype using the highly reliable and conserved matrix gene segment as the identification target.