Portable Multiplexed LATE-PCR Influenza Test

描述（由申请人提供）： 该项目将开发一种十重检测试剂盒，用于在采集原始患者样本后30分钟内检测表1所列的C类优先病原体流感。检测到的亚型都是H5和H7的高致病性菌株，它们被USDA归类为选择因子。该检测试剂盒将针对6色Bio-Seeqll开发，这是一种便携式、电池可操作的热循环仪/荧光计，设计有自动样品制备功能，便于在床旁临床市场使用。所要求的支持将加快流感检测的人类诊断版本的开发，并提供获得政府专业知识和资源的途径（例如，流感病毒株）。计划开展以下活动，以通过临床前验证推进检测试剂盒开发：1.通过迭代相互作用建模和经验验证，构建一种十重RT-LATE PCR检测试剂盒，在单管中检测任何临床相关流感品种（包括高致病性品种）。2.研究影响检测性能的因素：优化流感样本的RNA提取/纯化方案和冻干RT-PCR试剂配方，以获得最佳流感检测灵敏度、特异性和集成检测盒（USPD）的稳健性。3.对检测试剂盒进行临床前验证，包括与现有经验证的PCR检测试剂盒进行可比性检测。 表1. 检测目标和亚型鉴定。流感类型HA亚型NA亚型高与低致病性AH 1 N1、N2、其他 H3 N1、N2、其他 H5 N1、N2、其他 H7 N1、N2、其他是B型B HA 相关性：这种检测方法将能够更快、更有效地诊断流感，以检测疫情（特别是高致病性品种），减少不必要的抗生素使用，减少住院时间，并能够更有效地使用神经氨酸酶抑制剂，保护国家战略药物储备，以应对大流行性流感。

DESCRIPTION (provided by applicant): The project will develop a decaplex assay to detect varieties of the Category C Priority Pathogen, influenza, listed in Table 1, within 30 minutes of taking a raw patient sample. The subtypes that are detected are all highly pathogenic strains of H5 and H7, which are classed as Select Agents by the USDA. The assay will be developed for the 6-color Bio-Seeqll, a portable, battery operable, thermocycler / fluorometer designed with automated sample preparation for ease of use in the point-of-care clinical market. The requested support will accelerate the development of the human diagnostic version of the influenza assay, and provides a vehicle to gain access to government expertise and resources (e.g., influenza strains). The following activities are planned to advance assay development through pre-clinical validation: 1. Construct a decaplex RT-LATE PCR assay to detect of any of the clinically relevant influenza varieties (including highly pathogenic varieties), in a single tube through iterative interaction modeling and empirical verification. 2. Investigate factors affecting assay performance: Optimize RNA extraction / purification protocols for influenza samples, and lyophilized RT-PCR reagent formulation, for best influenza assay sensitivity, specificity, and robustness in the integrated cartridge (USPD). 3. Perform pre-clinical validation of assay, including comparability testing against available, validated PCR assays. Table 1. Assay targets and subtype identification.Influenza TypeHA SubtypeNA SubtypeHigh vs. Low PathogenicityAH1N1, N2, other H3N1, N2, other H5N1, N2, otherYes H7N1, N2, otherYesBType B HA Relevance: This assay will enable faster, more effective diagnosis of influenza to detect outbreaks (especially of highly pathogenic varieties), reduce unneeded antibiotic use, reduce hospital stays, and enable more effective use of neuraminidase inhibitors, preserving Strategic National Stockpile of drugs for response to pandemic influenza.