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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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Related in: MedlinePlus

Fluorescence emission imaging from the nuclemeters used for HIV viral load testing.The images are at 8, 24, 32, 40, 48 and 56 min after the start of incubation. The sample chambers connected to reaction-diffusion conduits 1, 2, 3 and 4 contained 104, 103, 102, and 0 (negative control) HIV-1 RNA templates.
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f3: Fluorescence emission imaging from the nuclemeters used for HIV viral load testing.The images are at 8, 24, 32, 40, 48 and 56 min after the start of incubation. The sample chambers connected to reaction-diffusion conduits 1, 2, 3 and 4 contained 104, 103, 102, and 0 (negative control) HIV-1 RNA templates.

Mentions: To demonstrate the performance of the nuclemeter, we used reverse transcription, loop-mediated isothermal amplification (RT-LAMP)2021 to quantify HIV viral load. Samples containing 0, 102, 103, and 104 HIV-1 RNA molecules were inserted into the four sample chambers (Fig. 1b) and incubated at 62.5°C using our custom-made, portable, processor (Fig. 2a). 0.04% (w/v) hydroxypropyl-methyl-cellulose (HPMC) was added to the RT-LAMP reaction mixture to slow amplicons' diffusion and obtain a well-defined reaction front (Supplementary Note 1). Upstream of the front, the amplification process had reached its conclusion due to depletion of reaction components, and the fluorescence emission intensity was nearly independent of target type and concentration. The emission from the reaction-diffusion conduits was monitored with the USB fluorescent microscope (Fig. 3 and Video 1). At any given time, the greater the number of target molecules, the larger XF. Thus, with appropriate calibration, the number of initial target molecules can be inferred from XF. Although XF increases as time increases at any target concentration, the differences between XF values associated with different concentrations are time-independent. In Fig. 3, we monitored fluoresce emission for nearly an hour. However, the information needed for viral load determination is available within less than 30 minutes.


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)

Fluorescence emission imaging from the nuclemeters used for HIV viral load testing.The images are at 8, 24, 32, 40, 48 and 56 min after the start of incubation. The sample chambers connected to reaction-diffusion conduits 1, 2, 3 and 4 contained 104, 103, 102, and 0 (negative control) HIV-1 RNA templates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Fluorescence emission imaging from the nuclemeters used for HIV viral load testing.The images are at 8, 24, 32, 40, 48 and 56 min after the start of incubation. The sample chambers connected to reaction-diffusion conduits 1, 2, 3 and 4 contained 104, 103, 102, and 0 (negative control) HIV-1 RNA templates.
Mentions: To demonstrate the performance of the nuclemeter, we used reverse transcription, loop-mediated isothermal amplification (RT-LAMP)2021 to quantify HIV viral load. Samples containing 0, 102, 103, and 104 HIV-1 RNA molecules were inserted into the four sample chambers (Fig. 1b) and incubated at 62.5°C using our custom-made, portable, processor (Fig. 2a). 0.04% (w/v) hydroxypropyl-methyl-cellulose (HPMC) was added to the RT-LAMP reaction mixture to slow amplicons' diffusion and obtain a well-defined reaction front (Supplementary Note 1). Upstream of the front, the amplification process had reached its conclusion due to depletion of reaction components, and the fluorescence emission intensity was nearly independent of target type and concentration. The emission from the reaction-diffusion conduits was monitored with the USB fluorescent microscope (Fig. 3 and Video 1). At any given time, the greater the number of target molecules, the larger XF. Thus, with appropriate calibration, the number of initial target molecules can be inferred from XF. Although XF increases as time increases at any target concentration, the differences between XF values associated with different concentrations are time-independent. In Fig. 3, we monitored fluoresce emission for nearly an hour. However, the information needed for viral load determination is available within less than 30 minutes.

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