Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on SlipChip.
Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on SlipChip.
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DOI：
10.1021/ac200247e
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发表时间：
2011-05-01
影响因子：
7.4
通讯作者：
Ismagilov, Rustem F.
中科院分区：
文献类型：
--
作者：
Shen, Feng;Davydova, Elena K.;Du, Wenbin;Kreutz, Jason E.;Piepenburg, Olaf;Ismagilov, Rustem F.
在本文中，通过化学引发超过一千个序列特异性的、纳升的、等温的平行扩增反应，在单分子水平上实现了核酸的数字定量检测。数字聚合酶链反应（数字PCR）是一种用于核酸定量的方法，可对单个分子扩增的存在或不存在进行计数。然而，它仍然需要温度循环，这在资源有限的条件下是不可取的。这使得核酸扩增的等温方法，如重组酶聚合酶扩增（RPA），更有吸引力。本文描述了一种微流体数字RPA滑动芯片，其用于通过在无仪器移液管加载后向每个反应室添加化学引发剂并通过简单的滑动步骤同时引发超过一千nL规模的RPA反应。两种设计的滑动芯片，两步滑动和一步滑动，使用数字RPA进行了验证。通过使用数字RPA滑动芯片，消除了孵育前RPA扩增反应预启动的假阳性结果。终点荧光读数用于“是或否”数字定量。通过扩增和计数靶核酸（耐甲氧西林金黄色葡萄球菌（MRSA）基因组DNA）的单分子，验证了滑动芯片中数字RPA的性能。SlipChip上的数字RPA还耐受孵育温度的波动（37-42 °C），并且其性能与相同SlipChip设计上的数字PCR相当。数字RPA SlipChip提供了一种简单的方法来定量核酸，而无需热循环或动力学测量，在资源有限的环境下具有诊断和环境监测的潜在应用。使用耐溶剂玻璃器件在纳升尺度上并行引发数千个化学反应的能力可能适用于更广泛的应用。
In this paper, digital quantitative detection of nucleic acids was achieved at the single-molecule level by chemical initiation of over one thousand sequence-specific, nanoliter, isothermal amplification reactions in parallel. Digital polymerase chain reaction (digital PCR), a method used for quantification of nucleic acids, counts the presence or absence of amplification of individual molecules. However it still requires temperature cycling, which is undesirable under resource-limited conditions. This makes isothermal methods for nucleic acid amplification, such as recombinase polymerase amplification (RPA), more attractive. A microfluidic digital RPA SlipChip is described here for simultaneous initiation of over one thousand nL-scale RPA reactions by adding a chemical initiator to each reaction compartment with a simple slipping step after instrument-free pipette loading. Two designs of the SlipChip, two-step slipping and one-step slipping, were validated using digital RPA. By using the digital RPA SlipChip, false positive results from pre-initiation of the RPA amplification reaction before incubation were eliminated. End-point fluorescence readout was used for “yes or no” digital quantification. The performance of digital RPA in a SlipChip was validated by amplifying and counting single molecules of the target nucleic acid, Methicillin-resistant Staphylococcus aureus (MRSA) genomic DNA. The digital RPA on SlipChip was also tolerant to fluctuations of the incubation temperature (37–42 °C), and its performance was comparable to digital PCR on the same SlipChip design. The digital RPA SlipChip provides a simple method to quantify nucleic acids without requiring thermal cycling or kinetic measurements, with potential applications in diagnostics and environmental monitoring under resource-limited settings. The ability to initiate thousands of chemical reactions in parallel on the nanoliter scale using solvent-resistant glass devices is likely to be useful for a broader range of applications.
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