Development or improvement of clinical diagnostic tests for SARS-CoV-2 to increase the sensitivity, specificity and ability to provide rapid results

摘要 未满足的需求：q-rtPCR技术已主导COVID-19诊断和公共卫生筛查。 独立于测试开发人员，q-rtPCR已被证明具有异常高的假阴性率： 15%至48%（1）。根据新冠病毒追踪项目。截至2020年5月16日，1100万次COVID-19检测 已在美国使用（2）。如果假阴性率为15%， 被错误地归类为无感染。正如所料，荟萃分析表明，假阴性 q-rtPCR的速率在感染的第7天之前"爆炸"（3），此时病毒载量仍然较低， 作为早期检测弱症状携带者的工具无效，同时也降低了其在 流行病学（4）。 解决方案：PathogenDx发明了一种基于微阵列的检测方法DetectX-Rv，并为其申请了专利， 已提交FDA-EUA审查，以筛查NP拭子中的COVID-19。微阵列有能力 根据FDA提交的要求，以[SARS-CoV-2]作为主要分析物，平行检测多种病毒分析物。 内容增强。我们在这里建议增加一个新发现的COVID-19进化枝变体， 被其他人假设为更具感染性（5）。DetectX-Rv已包含测试所需的内容 SARS-CoV-2+多种其他冠状病毒[SARS-CoV、MERS-CoV、CoV 229E、CoV OC 43、CoV NL 63、CoV HKU1]加流感+[PanA & PanB]，定义为"泛呼吸道"病毒检测集。 然而，对于本RO1中提出的开发，这种"额外"的冠状病毒内容将是合理的。 修改并用作大量特异性对照。本RO1以外的其他资金来源 将用于开发完整的泛呼吸道病毒内容，作为单独的产品。基于工作 到目前为止已经完成，包括2020年4月15日向FDA提交的申请，我们建议通过RO1资金， 变体（DetectX-Rv-v2）可以（在Q2前）准备好与NP拭子采集一起部署，作为自动化的 96阵列/SBS平板COVID-19检测（576次检测/班次）。 灵敏度提高。DetectX-Rv检测基于两个串联终点PCR反应 [富集+标记]结合微阵列杂交。这种技术方法可以在 单核苷酸分辨率超过6个对数的样品输入动态范围。最重要的是，[串联PCR + 杂交]测定通常产生最低检测限（LLOD）<1个基因组/反应。 我们预计，通过这种方法，我们将在以下条件下常规检测COVID-19（信号/噪音> 20 x背景） 每个反应只有一个病毒基因组。初步数据，包括提交给FDA EUA计划的数据，表明 DetectX-Rv的LLOD将比优化的q-RT-PCR反应低约10倍。因此， DetectX-Rv检测，COVID-19应常规检测100个病毒颗粒/拭子。 特异性增强。DetectX-Rv（144次测试）相对于q-rtPCR具有巨大的测试能力， 允许DetectX-Rv监测SARS-CoV-2基因组中的3个不同位点和人类RNA对照， 同时（N1、N2、N3、P）沿着与一组8个病毒对照平行进行，一式三份，因此 允许确认COVID-19信号，其实验特异性比q-rtPCR可达到的特异性高10倍。在 拟议的RO1计划，将对冗余的COVID-19内容进行修改（DetectX-Rv-v2），以包括 最近鉴定的进化枝变体S-D614G（5），使得可以同时测量初始和变体进化枝。 增强的功能。我们即将完成部署DetectX-Rv全自动化的计划 功能作为短期的RO1交付成果，我们将在2020年9月1日之前开发一套集成的技术 用于处理576个NP拭子或唾液测试/偏移，以96个微阵列/SBS板格式，基于劳动力 技术人员1名。96孔格式采用已经使用的相同印刷技术， （专利）微阵列制造化学，因此风险低。然而，通过完成向 96井格式，我们将实现12，000阵列/天的生产规模， 其他来源，可以立即扩大到每天48 000人。 根据PAR-20 - 178的要求，我们提出了4个具体目标来支持这一2年的RO1计划。 SA 1. Q1.添加进化枝和冠状病毒内容，使SARS-CoV-2和变体的分析成为可能。 SA 2. Q2. DetectX-Rv-v2与qRT-PCR实质等同器械的灵敏度/特异性分析：临床标本 SA 3. Q3 - 5.通过一锅RT-PCR和杂交率增强检测X-Rv-v2。 SA 4. Q5 - 8.增强和增强DetectX-Rv性能以诊断无症状传播

Abstract Unmet Need: q-rtPCR technology has dominated COVID-19 diagnostics and public health screening. Independent of the test developer, q-rtPCR has been shown to have an unusually high false negative rate: 15% up to 48% (1). According to the Covid Tracking Project. as of May 16th, 2020, 11 Million COVID-19 tests had been administered in the US (2). With 15% false negative rate, approximately 1.65M people would be falsely classified as free of infection. As might be expected, meta-analysis has shown that the false negative rate for q-rtPCR “explodes” before day 7 of infection (3) when viral load is still low, to render q-rtPCR ineffective as a tool for detecting weakly symptomatic carriers early, while also lessening its value in epidemiology (4). The Solution: PathogenDx has invented, patented and developed a microarray-based test, DetectX-Rv, and has submitted it for FDA-EUA review to screen for COVID-19 in NP swabs. The microarray has the capacity to test for multiple viral analytes in parallel with [SARS-CoV-2] as the primary analyte under FDA submission. Content Enhancement. We propose here the addition of a newly identified COVID-19 clade variant, which has been hypothesized by others to be more infective (5). DetectX-Rv already contains content needed to test SARS-CoV-2 plus multiple other coronavirus [SARS-CoV, MERS-CoV, CoV 229E,CoV OC43, CoV NL63, CoV HKU1] plus influenza + [PanA & Pan B] which are defined as a set as a “Pan-Respiratory” Virus Test. However for the development proposed in this RO1, this “extra” coronavirus content will be rationally modified and used instead as a large set of specificity controls. Other sources of funding outside this RO1 will be used to develop the full Pan-Respiratory virus content, as a separate product. Based on the work completed thus far, including April 15, 2020 filing to the FDA, we propose that with RO1 funding, the new test variant (DetectX-Rv-v2) can be made ready (by Q2) for deployment with NP swab collection as an automated 96 array/SBS plate COVID-19 test (@576 tests/shift). Sensitivity Improvement. The DetectX-Rv test is based on two Tandem Endpoint PCR reactions in series [Enrichment + Labeling] coupled to microarray hybridization. This technical approach gives rise to detection at single nucleotide resolution over a 6-log sample input dynamic range. Most importantly, the [Tandem PCR + Hybridization] assay routinely generates a Lowest Limit of Detection (LLOD) <1 genome per reaction. We anticipate that with this approach, we will routinely detect COVID-19 (signal/noise >20x background) at only 1 viral genome per reaction. Preliminary data, including that submitted to the FDA EUA program, suggests that the LLOD for DetectX-Rv will be roughly 10x lower than for an optimized q-RT-PCR reaction. Thus, with the DetectX-Rv test, COVID-19 should be routinely detected at 100 virus particles/swab. Specificity Enhancement. DetectX-Rv (144 tests) has enormous test capacity relative to q-rtPCR, which has allowed DetectX-Rv to monitor 3 different sites in the SARS-CoV-2 genome and a human RNA control, concurrently (N1,N2,N3,P) along with a panel of 8 viral controls all performed in parallel and in triplicate, thus allowing confirmation of COVID-19 signals with experimental specificity >10x than attainable with q-rtPCR. In the proposed RO1 program, that redundant COVID-19 content will be amended (DetectX-Rv-v2) to include a recently-identified clade variant S-D614G (5) so that initial and variant clades can be measured concurrently. Throughput Enhancement. We are near completion of a program to deploy full automation of DetectX-Rv function. As a short term RO1 deliverable, we will develop, by Sept 1, 2020, an integrated suite of technology for processing 576 NP swab or saliva tests/shift, in a 96 microarray/SBS plate format, based on the labor burden of 1 technician. The 96 well format employs the same printing technology already in use and the same (patented) microarray fabrication chemistry and is thus low risk. However, by completing the transition to the 96 well format, we will have achieved 12,000 arrays/day manufacturing scale, which with support from equity other sources, could be immediately scaled to 48,000 per day. Based the requirements of PAR-20-178, we propose 4 Specific Aims to support this 2 year RO1 program. SA1. Q1. Add Clade & Coronavirus Content Enabling Analysis of SARS-CoV-2 & variants. SA2. Q2. Sensitivity/specificity analysis of DetectX-Rv-v2 vs qRT-PCR predicate: Clinical Specimens SA3. Q3-5. Simplify DetectX-Rv-v2 Throughput via One pot RT-PCR & Hybridization Rate Enhancement. SA4. Q5-8. Enhance & Validate DetectX-Rv Performance to Diagnose Asymptomatic Transmission