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

用于低拷贝数核酸绝对定量的高通量微滴数字 PCR 平台

• 批准号: RTI-2019-00410
• 负责人: Vaillancourt, Cathy
• 金额: $ 10.33万
• 依托单位: Institut national de la recherche scientifique
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=667431
• 关键词: HighThroughput; Droplet; Digital; PCR; Platform

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.

进行液滴数字PCR （ddPCR）分析的能力对于申请人的NSERC-DG研究项目至关重要。申请人不能继续使用“传统”qPCR技术定量mRNA，微生物和功能基因，并且任何延迟获得ddPCR系统平台将妨碍他们在NSERC研究计划框架内产生最高质量的定量结果。虽然实时PCR （qPCR）可以用于各种各样的应用，但ddPCR是许多人更好的选择。由于样本划分和绝对数字定量（而不是基于标准曲线的相对定量），ddPCR最适合要求高精度、准确性、灵敏度和重复性高的应用。由于ddPCR是通过计数阳性液滴作为终点绝对定量进行的，扩增效率不像qPCR那样是一个重要的因素，也不需要测定特定的标准曲线，节省了大量的时间和金钱，同时防止了结果的偏差。这种独特的技术完全满足了对内部绝对核酸定量系统的迫切需求，这将补充申请人的实时PCR平台，并使他们能够进行绝对准确的定量核酸分析。该系统对提高我院环境毒理学课题组的研究水平和工作效率具有重要意义。（co）申请人的不同研究活动包括低丰度RNA的分析；病原体检测;病毒载量检测；miRNA分析和下一代测序样本定量（所有申请人），以及受环境因素影响的植物微生物群（Yergeau）、致病菌（dsamziel）、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，所有申请人的研究都依赖于他们所研究的基质（组织、细菌、土壤）中核酸低拷贝数的准确可靠的定量，因此他们依赖于ddPCR系统。此外，ddPCR系统平台的可用性将对HQP的研究项目产生直接影响，促进在高影响力期刊上的发表，并使HQP在追求研究生涯中更具竞争力。延迟采购所要求的设备将对上述所有研究方案的进展产生巨大的负面影响，并将阻止它们在批准期内完成。