title: Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic Acids.
abstract: Absolute, precise quantification methods expand the scope of nucleic acids research and have many practical applications. Digital polymerase chain reaction (dPCR) is a powerful method for nucleic acid detection and absolute quantification. However, it requires thermal cycling and accurate temperature control, which are difficult in resource-limited conditions. Accordingly, isothermal methods, such as recombinase polymerase amplification (RPA), are more attractive. We developed a picoliter well array (PWA) chip with 27,000 consistently sized picoliter reactions (314 pL) for isothermal DNA quantification using digital RPA (dRPA) at 39°C. Sample loading using a scraping liquid blade was simple, fast, and required small reagent volumes (i.e., <20 μL). Passivating the chip surface using a methoxy-PEG-silane agent effectively eliminated cross-contamination during dRPA. Our creative optical design enabled wide-field fluorescence imaging in situ and both end-point and real-time analyses of picoliter wells in a 6-cm(2) area. It was not necessary to use scan shooting and stitch serial small images together. Using this method, we quantified serial dilutions of a Listeria monocytogenes gDNA stock solution from 9 × 10(-1) to 4 × 10(-3) copies per well with an average error of less than 11% (N = 15). Overall dRPA-on-chip processing required less than 30 min, which was a 4-fold decrease compared to dPCR, requiring approximately 2 h. dRPA on the PWA chip provides a simple and highly sensitive method to quantify nucleic acids without thermal cycling or precise micropump/microvalve control. It has applications in fast field analysis and critical clinical diagnostics under resource-limited settings.
authors.affiliation: State Key Laboratory on Integrated Optoelectronics, Institute of Semiconductors, Chinese Academy of Sciences, P.O. Box 912, Beijing, 100083, China.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Li
authors.affiliation: State Key Laboratory on Integrated Optoelectronics, Institute of Semiconductors, Chinese Academy of Sciences, P.O. Box 912, Beijing, 100083, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: State Key Laboratory on Integrated Optoelectronics, Institute of Semiconductors, Chinese Academy of Sciences, P.O. Box 912, Beijing, 100083, China.
authors.forename: Qingquan
authors.initials: Q
authors.lastname: Wei
authors.affiliation: State Key Laboratory on Integrated Optoelectronics, Institute of Semiconductors, Chinese Academy of Sciences, P.O. Box 912, Beijing, 100083, China.
authors.forename: Yuanjie
authors.initials: Y
authors.lastname: Liu
authors.affiliation: State Key Laboratory on Integrated Optoelectronics, Institute of Semiconductors, Chinese Academy of Sciences, P.O. Box 912, Beijing, 100083, China.
authors.forename: Wenwen
authors.initials: W
authors.lastname: Liu
authors.affiliation: State Key Laboratory on Integrated Optoelectronics, Institute of Semiconductors, Chinese Academy of Sciences, P.O. Box 912, Beijing, 100083, China.
authors.forename: Xuelian
authors.initials: X
authors.lastname: Zhang
authors.affiliation: State Key Laboratory on Integrated Optoelectronics, Institute of Semiconductors, Chinese Academy of Sciences, P.O. Box 912, Beijing, 100083, China.|Joint Laboratory of Bioinformation Acquisition and Sensing Technology, Institute of Semiconductors, Beijing Institute of Genomics, Chinese Academy of Sciences, P.O. Box 912, Beijing 100083, China.
authors.forename: Yude
authors.initials: Y
authors.lastname: Yu
mesh_terms: D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D045522:Recombinases; D013696:Temperature
chemical_list: D045522:Recombinases