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Nuclemeter: a reaction-diffusion based method for quantifying nucleic acids undergoing enzymatic amplification.

Liu C, Sadik MM, Mauk MG, Edelstein PH, Bushman FD, Gross R, Bau HH - Sci Rep (2014)

Bottom Line: Typically, nucleic acid quantification requires expensive instruments, such as real-time PCR machines, which are not appropriate for on-site use and for low-resource settings.The number of target molecules is inferred from the position of the reaction-diffusion front, analogous to reading temperature in a mercury thermometer.The proposed method is suitable for nucleic acid quantification at point of care, compatible with multiplexing and high-throughput processing, and can function instrument-free.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical Engineering and Applied Mechanics, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Real-time amplification and quantification of specific nucleic acid sequences plays a major role in medical and biotechnological applications. In the case of infectious diseases, such as HIV, quantification of the pathogen-load in patient specimens is critical to assess disease progression and effectiveness of drug therapy. Typically, nucleic acid quantification requires expensive instruments, such as real-time PCR machines, which are not appropriate for on-site use and for low-resource settings. This paper describes a simple, low-cost, reaction-diffusion based method for end-point quantification of target nucleic acids undergoing enzymatic amplification. The number of target molecules is inferred from the position of the reaction-diffusion front, analogous to reading temperature in a mercury thermometer. The method was tested for HIV viral load monitoring and performed on par with conventional benchtop methods. The proposed method is suitable for nucleic acid quantification at point of care, compatible with multiplexing and high-throughput processing, and can function instrument-free.

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The reproducibility and sensitivity of the nuclemeter.(a) Four nuclemeters each containing identical target concentrations (102 copies HIV-1 RNA) to illustrate reproducibility. (b) Evaluation of the limits of detection of the nuclemeter. Sample chambers connected to reaction-diffusion conduits 1, 2, 3 and 4 contain, respectively, 50, 50, 5 and 5 copies of HIV RNA target.
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f4: The reproducibility and sensitivity of the nuclemeter.(a) Four nuclemeters each containing identical target concentrations (102 copies HIV-1 RNA) to illustrate reproducibility. (b) Evaluation of the limits of detection of the nuclemeter. Sample chambers connected to reaction-diffusion conduits 1, 2, 3 and 4 contain, respectively, 50, 50, 5 and 5 copies of HIV RNA target.

Mentions: The reproducibility of the nuclemeter was evaluated by introducing identical target concentrations (102 copies of HIV-1 RNA) into all four sample chambers (Fig. 4a). All four conduits exhibited nearly identical length emission columns XF (±4.5%) at any given time.


Nuclemeter: a reaction-diffusion based method for quantifying nucleic acids undergoing enzymatic amplification.

Liu C, Sadik MM, Mauk MG, Edelstein PH, Bushman FD, Gross R, Bau HH - Sci Rep (2014)

The reproducibility and sensitivity of the nuclemeter.(a) Four nuclemeters each containing identical target concentrations (102 copies HIV-1 RNA) to illustrate reproducibility. (b) Evaluation of the limits of detection of the nuclemeter. Sample chambers connected to reaction-diffusion conduits 1, 2, 3 and 4 contain, respectively, 50, 50, 5 and 5 copies of HIV RNA target.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256561&req=5

f4: The reproducibility and sensitivity of the nuclemeter.(a) Four nuclemeters each containing identical target concentrations (102 copies HIV-1 RNA) to illustrate reproducibility. (b) Evaluation of the limits of detection of the nuclemeter. Sample chambers connected to reaction-diffusion conduits 1, 2, 3 and 4 contain, respectively, 50, 50, 5 and 5 copies of HIV RNA target.
Mentions: The reproducibility of the nuclemeter was evaluated by introducing identical target concentrations (102 copies of HIV-1 RNA) into all four sample chambers (Fig. 4a). All four conduits exhibited nearly identical length emission columns XF (±4.5%) at any given time.

Bottom Line: Typically, nucleic acid quantification requires expensive instruments, such as real-time PCR machines, which are not appropriate for on-site use and for low-resource settings.The number of target molecules is inferred from the position of the reaction-diffusion front, analogous to reading temperature in a mercury thermometer.The proposed method is suitable for nucleic acid quantification at point of care, compatible with multiplexing and high-throughput processing, and can function instrument-free.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical Engineering and Applied Mechanics, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Real-time amplification and quantification of specific nucleic acid sequences plays a major role in medical and biotechnological applications. In the case of infectious diseases, such as HIV, quantification of the pathogen-load in patient specimens is critical to assess disease progression and effectiveness of drug therapy. Typically, nucleic acid quantification requires expensive instruments, such as real-time PCR machines, which are not appropriate for on-site use and for low-resource settings. This paper describes a simple, low-cost, reaction-diffusion based method for end-point quantification of target nucleic acids undergoing enzymatic amplification. The number of target molecules is inferred from the position of the reaction-diffusion front, analogous to reading temperature in a mercury thermometer. The method was tested for HIV viral load monitoring and performed on par with conventional benchtop methods. The proposed method is suitable for nucleic acid quantification at point of care, compatible with multiplexing and high-throughput processing, and can function instrument-free.

Show MeSH
Related in: MedlinePlus