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，用于快速和同时筛选甲型和乙型流感及其重要亚型的临床样本。与现有的流感病毒聚合酶链式反应(PCR)方法相比，该方法的优势来自于使用单个基因片段来识别类型和亚型。这项拟议工作的靶点是基质基因片段，众所周知，它比传统的血凝素基因(HA)靶标扩增得很好，而且更保守。开发的产品最初的商业利基市场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收用于分型和分型的临床样本，但按照病毒分离和血凝抑制试验的“黄金标准”进行分型的成本令人望而却步。目前，大多数卫生实验室依靠基于荧光的免疫分析来筛选H3或H1病毒，没有确认神经氨酸酶亚型，也没有目前对新出现病毒的能力。那些使用实时逆转录聚合酶链式反应(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析，并且必须对每个HA亚型进行单独的检测。拟议的产品将作为一种手段，通过提供一种快速和具有成本效益的手段，同时筛查(A和B型)和某些亚型，特别是当前人类适应的流感病毒(A/H3N2和A/H1N1)和A/H5N1，从而改善和扩大美国州和地方一级以及世界各地区域实验室的监测工作。这一新的监测工具将取代现有的免疫分析和基于单链HA靶向RT-PCR的分析，但不是为了取代病毒分离方法和测序，后者是更全面地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。要检验的假设是，可以设计一小组用于RT-PCR的M基因片段特异性引物对(5)，以选择性地检测A/H3N1、A/H1N1、A/H5N1和B病毒。具体目标2将重点放在RT-PCR多重条件的系统优化上。在具体目标3中，满足初始样本成功标准的有希望的引物组将在至少两个流感季节通过各种采样方法(包括鼻拭子、鼻腔冲洗和鼻咽抽吸)获得的300多名患者样本的盲法研究中得到验证。在第二阶段，我们将设计一个自动样本处理系统，包括提取和RT-PCR扩增，然后通过快速层析方法快速分离和检测PCR产物。与多重RRT-PCR相比，后PCR分离和检测有望提供更高的准确度和灵敏度，而不会增加整个分析的时间。 这项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，以及需要快速、廉价的病毒株监测工具。其目的是使州和地方公共卫生实验室能够使用高度可靠和保守的基质基因片段作为鉴定目标，以负担得起的价格快速筛查患者样本的流感类型和亚型。

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.