HighThroughput Droplet Digital PCR Platform for Absolute Quantitation of Nucleic Acids Low Copy Number

进行水滴数字聚合酶链式反应(DdPCR)分析的能力对申请者的NSERC-DG研究计划至关重要。申请者不能继续使用“传统的”定量聚合酶链式反应技术对信使核糖核酸、微生物和功能基因进行量化，而且在获得ddPCR系统平台方面的任何延误都将阻碍他们产生在他们的NSERC研究计划框架内预期的最高质量的定量结果。虽然实时聚合酶链式反应(QPCR)可用于多种应用，但对许多应用而言，ddPCR是更好的选择。由于样本分割和绝对数字定量(而不是基于标准曲线的相对定量)，ddPCR最适合于要求高精密度、准确度、灵敏度和重复性的应用。由于ddPCR是通过计数阳性液滴作为终点进行绝对定量的，因此扩增效率不像qPCR那样重要，也不需要特定于检测的标准曲线，从而节省了大量的时间和金钱，同时防止了结果的扭曲。这项独特的技术完美地满足了对内部绝对核酸定量系统的迫切需求，该系统将补充申请者的实时聚合酶链式反应平台，并使他们能够进行绝对准确的核酸定量分析。这个系统对于我们研究所Armand Frappier研究所环境毒理学研究小组的进步和提高效率是必要的。(共同)申请者的各种研究活动包括低丰度RNA分析；病原体检测；病毒载量检测；miRNA分析和下一代测序样本量化(所有申请者)，以及受环境因素影响的植物微生物区系(Yergeau)、病原菌(Déziel)、炎症和免疫生物标志物(Bernier)、水污染(Villemur)、类固醇生成酶(Sanderson)和胎盘功能(Vaillancourt)的测量。因此，申请者的所有研究都依赖于对其研究基质(组织、细菌、土壤)中低拷贝数核酸的准确和可靠的量化，因此他们依赖于ddPCR系统。此外，ddPCR系统平台的提供将对HQP研究项目产生直接影响，促进在影响力较大的期刊上发表文章，并使HQP在追求研究事业方面更具竞争力。延迟购置所需设备将对上述所有研究方案的进展产生重大负面影响，并将使其无法在授权期内完成。

The capability of performing droplet digital PCR (ddPCR) analyses is vital for the NSERC-DG research programs of the applicants. The applicants cannot continue quantifying mRNA, microorganisms and functional genes using "conventional" qPCR technology, and any delays in getting the ddPCR system platform will hamper them from producing the highest quality quantitative results that are expected in the frame of their NSERC research programs. Although real-time PCR (qPCR) can be used for a wide variety of applications, ddPCR is a better choice for many. Due to sample partitioning and absolute digital quantification (instead of standard curve-based relative quantification), ddPCR is best suited for applications requiring high precision, accuracy, sensitivity and reproducibility. Since ddPCR is performed as an endpoint absolute quantification by counting positive droplets, amplification efficiency is not a factor as important as in qPCR and there is no need for an assay-specific standard curve, saving a lot of time and money while preventing skewed results. This unique technology perfectly matches the urgent needs for an in-house absolute nucleic acids quantification system, which will complement the applicants' real-time PCR platforms and will allow them to conduct absolute accurate quantitative nucleic acids analyses. This system is necessary for the advancement and increased efficiency of the environmental toxicology research group at our institute INRS-Institut Armand Frappier. The diverse research activities of the (co)applicants cover the analysis of low-abundance RNA; pathogen detection; viral load detection; miRNA analysis and next generation sequencing sample quantification (all applicants) as well as measurement of plant microbiota (Yergeau), pathogenic bacteria (Déziel), inflammatory and immunologic biomarkers (Bernier), water contamination (Villemur), steroidogenic enzyme (Sanderson) and placental functions (Vaillancourt) affected by environmental factors. Thus, all the applicants' research relies on accurate and reliable quantification of low copy number of nucleic acids in their studied matrices (tissues, bacteria, soil), and thus they are dependent on ddPCR systems. Furthermore, availability of the ddPCR system platform will have a direct impact on HQP research projects, facilitating publication in high-impact journals, and making HQP more competitive in pursuing a research career. A delay in the acquisition of the requested equipment would have a dramatic negative impact on the progress of all the aforementioned research programs and would prevent their completion within the granting period.