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流感a型和B型及重要亚型的临床样本。与现有的流感病毒聚合酶链反应（PCR）方法相比，所提出的方法的优势在于使用单个基因片段来识别流感病毒的型和亚型。这项工作的目标，即基质基因片段，已知比传统的血凝素基因（HA）目标扩增得更强，更保守。开发的产品最初的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分型，但按照病毒分离和血凝抑制试验的“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测定来筛选H3或H1病毒，没有神经氨酸酶亚型的确认，目前也没有能力检测新出现的病毒。那些利用实时逆转录PCR （RRT-PCR）检测的实验室必须依赖易突变的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，同时筛查型（a和B）和某些亚型，特别是当前的人类适应型流感病毒（a /H3N2和a /H1N1）和a /H5N1，改善和扩大美国州和地方各级以及全球区域实验室的监测工作。这种新的监测工具将用于取代现有的免疫测定法和基于单一HA靶向RT-PCR的测定法，但其设计目的不是为了取代病毒分离方法和测序，而这是更全面地了解流感病毒所必需的。

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.