基于具有交叉反应活性探针的痕量核酸定量

An Absolute Quantitation of Minute DNA Templates by Cross-reactive Probes

数字PCR(digital PCR,dPCR)相比传统定量PCR具有多种优势,然而,其终点法的思路及单分子扩增体系需要对实验进行更细致的优化。数字PCR最低出版规范(the Minimum Information for the Publication of Digital PCR Experiments,d MIQE)提出了优化思路。然而,大多数发表的论文没有实现dMIQE的优化思路。本研究拟将利用探针的交叉扩增活性来实现数字PCR的优化。痕量DNA模板的检测以及定量是分子生物学中的重要应用之一。以结肠癌相关转录因子2(colon cancer correlation transcription factor 2,CCAT2)基因的G/T片段来模拟痕量核酸,并以梯度浓度的模板作为内参照来指示非特异扩增,进行微滴式数字PCR(droplet digital PCR,ddPCR)。首先,以qPCR方法确认探针的交叉扩增活性,并通过梯度PCR方法确认最佳区分温度(59℃),用于随后的实验。在梯度模板对照指引下,针对非特异扩增做出设置。本研究通过应用具有交叉扩增活性的TaqMan~探针,以微滴式数字PCR技术,在不同通道以高低活性交叉确定痕量的PCR模板(T片段为6.8 copies/μL;G片段为4.2 copies/μL;总量为11 copies/μL),实现了交叉验证。本研究提出的以高低活性的扩增实现互相验证的策略,可以作为dMIQE的优化方案,并提高ddPCR的可信度。

Digital PCR（ dPCR） offers several advantages over traditional real-time quantitative PCR. As relies on amplification from a single template by end-point fluorescence,the Minimum Information for the Publication of Digital PCR Experiments（ dMIQE） recommends that dPCR needs much deeper optimization. However,few current dPCR publications followed the recommendations. Here we used probes with cross-reactivity and performed droplet digital PCR（ ddPCR）,thus providing a strategy for dPCR optimization following the dMIQE guide. In this study,ddPCR was performed to quantify the raresequence detection with cross-reactivity probes. The G or T allelic gene fragments of CCAT2 served as the template to mimic the minute sequence and the gradient dilution of allelic fragment templates were added to demonstrate the nonspecific amplitude as reference controls. The probe for rs6983267 from Thermo Fisher showed cross-reactivity. Through temperature gradients optimization,59℃ was selected for subsequent experiments. By performing ddPCR with the setting for nonspecific amplification directed by gradient templates,we quantified the minute sequence under the different settings especially for the nonspecific amplification（ the concentration of T-allelic fragments is 6. 8 copies/μL; the concentration of G-allelic fragments is 4. 2 copies/μL; total concentration is 11 copies/μL）. Importantly, the concentration results of the minute template involved was confirmed by high and low fluorescence signal from different channels. The strategy we described here partly fulfills the dMIQE recommendation and offers more confidence of ddPCR.