Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic AcidsReport as inadecuate




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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-cm2 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.



Author: Zhao Li, Yong Liu, Qingquan Wei, Yuanjie Liu, Wenwen Liu, Xuelian Zhang, Yude Yu

Source: http://plos.srce.hr/



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