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Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on SlipChip.

ABSTRACT: 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 pipet 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 preinitiation 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.

SUBMITTER: Shen F

PROVIDER: S-EPMC3101872 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on SlipChip.

Shen Feng F Davydova Elena K EK Du Wenbin W Kreutz Jason E JE Piepenburg Olaf O Ismagilov Rustem F RF

Analytical chemistry 20110408 9

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 condition ...[more]

PMID: 21476587

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Project description:Bacterial infection is a widespread problem in humans that can potentially lead to hospitalization and morbidity. The largest obstacle for physicians/clinicians is the time delay in accurately identifying infectious bacteria, especially their sub-species, in order to adequately treat and diagnose such infected patients. Loop-mediated amplification (LAMP) is a nucleic acid amplification method that has been widely used in diagnostic applications due to its simplicity of constant temperature, use of up to 4 to 6 primers (rendering it highly specific), and capability of amplifying low copies of target sequences. Use of interfacial effect-based monitoring is expected to dramatically shorten the time-to-results of nucleic acid amplification techniques. In this work, we developed a LAMP-based point-of-care platform for detection of bacterial infection, utilizing smartphone measurement of contact angle from oil-immersed droplet LAMP reactions. Whole bacteria (Escherichia coli O157:H7) were assayed in buffer as well as 5% diluted human whole blood. Monitoring of droplet LAMP reactions was demonstrated in a three-compartment, isothermal proportional-integrated-derived (PID)-controlled chip. Smartphone-captured images of droplet LAMP reactions, and their contact angles, were evaluated. Contact angle decreased substantially upon target amplification in both buffer and whole blood samples. In comparison, no-target control (NTC) droplets remained stable throughout the 30?min isothermal reactions. These results were explained by the pre-adsorption of plasma proteins to an oil-water interface (lowering contact angle), followed by time-dependent amplicon formation and their preferential adsorption to the plasma protein-occupied oil-water interface. Time-to-results was as fast as 5?min, allowing physicians to quickly make their decision for infected patients. The developed assay demonstrated quantification of bacteria concentration, with a limit-of-detection at 102?CFU/?L for buffer samples, and binary target or no-target identification with a limit-of-detection at 10?CFU/?L for 5% diluted whole blood samples.

Dataset Information

Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on SlipChip.

Publications

Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on SlipChip.

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