Screening of Additive Formulations Enables Off-Chip Drop Reverse Transcription Quantitative Polymerase Chain Reaction of Single Influenza A Virus Genomes
Screening of Additive Formulations Enables Off-Chip Drop Reverse Transcription Quantitative Polymerase Chain Reaction of Single Influenza A Virus Genomes
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DOI：
10.1021/acs.analchem.0c03455
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发表时间：
2021-02-26
影响因子：
7.4
通讯作者：
Chang, Connie B.
中科院分区：
文献类型：
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作者：
Loveday, Emma Kate;Zath, Geoffrey K.;Chang, Connie B.
使用基于液滴的微流控技术的聚合酶链式反应(PCR)的小型化允许在皮升大小的水滴中扩增单个核酸。聚合酶链式反应过程中准确的数据收集要求液滴在热循环过程中保持稳定并保持液滴含量不变。在对已知的聚合酶链式反应添加剂进行系统测试后，我们确定了最佳配方为：1%w/v吐温-20，0.8微克/亩L牛血清白蛋白，1 M甜菜碱在水相中，以及3wt%(w/w)的聚乙二醇全氟聚醚(2)表面活性剂在直径50微米的油相中，保持液滴稳定性并防止染料迁移。这种配方实现了一种我们称为芯片外液滴反转录定量聚合酶链式反应(OCD RT-qPCR)的方法，在这种方法中，液滴在qPCR机中进行热循环，并以不同的循环次数在芯片外或微流控芯片外进行采样。使用OCD RT-qPCR从数百个单独的液滴构建并使用荧光显微镜成像的qPCR扩增曲线与使用qPCR机进行热循环的类似300,000液滴的扩增曲线相关。为了证明强迫症RT-qPCR的实用性，甲型流感病毒的核糖核酸被检测到每滴只有一个病毒基因组拷贝，即0.320个CPD。这项工作扩展到对滴状病毒中的IAV M基因RNA和细胞β-肌动蛋白DNA进行多重检测，并直接从感染细胞中扩增IAV基因组，而不需要单独的RNA提取步骤。优化的添加剂配方和OCD-qPCR方法允许在没有复杂设备的情况下进行基于液滴的RT-qPCR，并展示了以最少的处理对液滴中单个或稀有核酸物种进行量化的能力。
The miniaturization of polymerase chain reaction (PCR) using drop-based microfluidics allows for amplification of single nucleic acids in aqueous picoliter-sized drops. Accurate data collection during PCR requires that drops remain stable to coalescence during thermocycling and drop contents are retained. Following systematic testing of known PCR additives, we identified an optimized formulation of 1% w/v Tween-20, 0.8 mu g/mu L bovine serum albumin, 1 M betaine in the aqueous phase, and 3 wt % (w/w) of the polyethylene glycol-perfluoropolyether(2) surfactant in the oil phase of 50 mu m diameter drops that maintains drop stability and prevents dye transport. This formulation enables a method we call off-chip drop reverse transcription quantitative PCR (OCD RT-qPCR) in which drops are thermocycled in a qPCR machine and sampled at various cycle numbers "off-chip", or outside of a microfluidic chip. qPCR amplification curves constructed from hundreds of individual drops using OCD RT-qPCR and imaged using epifluorescence microscopy correlate with amplification curves of similar to 300,000 drops thermocycled using a qPCR machine. To demonstrate the utility of OCD RT-qPCR, influenza A virus (IAV) RNA was detected down to a single viral genome copy per drop, or 0.320 cpd. This work was extended to perform multiplexed detection of IAV M gene RNA and cellular beta-actin DNA in drops, and direct amplification of IAV genomes from infected cells without a separate RNA extraction step. The optimized additive formulation and the OCD-qPCR method allow for drop-based RT-qPCR without complex devices and demonstrate the ability to quantify individual or rare nucleic acid species within drops with minimal processing.