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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

Experimental data and theoretical predictions of nuclemeter's performance.(a) Normalized emission intensity  as a function of position along the reaction-diffusion conduit at various times. The solid lines and symbols correspond, respectively, to predictions and experimental data. The number of target molecules is 103 copies. (b) Normalized emission intensity  as a function of time at positions x = 1.2, 1.8, and 2.4 mm along the length of the conduit. The solid lines and symbols correspond, respectively, to the predictions and experimental data. The number of target molecules is 103 copies. (c) The experimental rate of the reaction  as a function of position (x) at various times. (d) The measured width of the reaction-rate peak at midheight Λexp as a function of time. (e) The measured position of the reaction front XF, exp as a function of time for various template concentrations (error bars = s.d.; n = 3; R2 = 0.998). (f) The intercept (t0, exp) of the line in Fig. 5e and the threshold time Ct of real time, benchtop RT-LAMP curves as functions of the number of templates (error bars = s.d.; n = 3; R2 = 0.99). (g) XF, exp-XF,exp(3) as a function of the template number at various times t (error bars = s.d.; R2 = 0.99, n = 15). (h) The predicted position of the reaction front (XF, th) as a function of time for various numbers of templates. (i) The predicted intercept (t0, th) of the asymptotes in Fig. 5h as a function of template number.
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f5: Experimental data and theoretical predictions of nuclemeter's performance.(a) Normalized emission intensity as a function of position along the reaction-diffusion conduit at various times. The solid lines and symbols correspond, respectively, to predictions and experimental data. The number of target molecules is 103 copies. (b) Normalized emission intensity as a function of time at positions x = 1.2, 1.8, and 2.4 mm along the length of the conduit. The solid lines and symbols correspond, respectively, to the predictions and experimental data. The number of target molecules is 103 copies. (c) The experimental rate of the reaction as a function of position (x) at various times. (d) The measured width of the reaction-rate peak at midheight Λexp as a function of time. (e) The measured position of the reaction front XF, exp as a function of time for various template concentrations (error bars = s.d.; n = 3; R2 = 0.998). (f) The intercept (t0, exp) of the line in Fig. 5e and the threshold time Ct of real time, benchtop RT-LAMP curves as functions of the number of templates (error bars = s.d.; n = 3; R2 = 0.99). (g) XF, exp-XF,exp(3) as a function of the template number at various times t (error bars = s.d.; R2 = 0.99, n = 15). (h) The predicted position of the reaction front (XF, th) as a function of time for various numbers of templates. (i) The predicted intercept (t0, th) of the asymptotes in Fig. 5h as a function of template number.

Mentions: Fig. 5 analyzes the experimental data and compares it with the predictions of a simple theoretical model (to be described later). We take the emission intensity to be proportional to the amplicons' concentration c(x,t), assumed uniform in each cross-section of the conduit. Fig. 5a depicts as a function of position x at various times t. The lines and symbols correspond, respectively, to predictions and experimental data. We define the location of the reaction front XF(t) as the position at which . When x < XF, and the amplification reaction is nearly complete (the bright regions with fluorescent emission in Fig. 3). When x > XF, and no amplification has yet occurred (the dark regions in Fig. 3).


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)

Experimental data and theoretical predictions of nuclemeter's performance.(a) Normalized emission intensity  as a function of position along the reaction-diffusion conduit at various times. The solid lines and symbols correspond, respectively, to predictions and experimental data. The number of target molecules is 103 copies. (b) Normalized emission intensity  as a function of time at positions x = 1.2, 1.8, and 2.4 mm along the length of the conduit. The solid lines and symbols correspond, respectively, to the predictions and experimental data. The number of target molecules is 103 copies. (c) The experimental rate of the reaction  as a function of position (x) at various times. (d) The measured width of the reaction-rate peak at midheight Λexp as a function of time. (e) The measured position of the reaction front XF, exp as a function of time for various template concentrations (error bars = s.d.; n = 3; R2 = 0.998). (f) The intercept (t0, exp) of the line in Fig. 5e and the threshold time Ct of real time, benchtop RT-LAMP curves as functions of the number of templates (error bars = s.d.; n = 3; R2 = 0.99). (g) XF, exp-XF,exp(3) as a function of the template number at various times t (error bars = s.d.; R2 = 0.99, n = 15). (h) The predicted position of the reaction front (XF, th) as a function of time for various numbers of templates. (i) The predicted intercept (t0, th) of the asymptotes in Fig. 5h as a function of template number.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Experimental data and theoretical predictions of nuclemeter's performance.(a) Normalized emission intensity as a function of position along the reaction-diffusion conduit at various times. The solid lines and symbols correspond, respectively, to predictions and experimental data. The number of target molecules is 103 copies. (b) Normalized emission intensity as a function of time at positions x = 1.2, 1.8, and 2.4 mm along the length of the conduit. The solid lines and symbols correspond, respectively, to the predictions and experimental data. The number of target molecules is 103 copies. (c) The experimental rate of the reaction as a function of position (x) at various times. (d) The measured width of the reaction-rate peak at midheight Λexp as a function of time. (e) The measured position of the reaction front XF, exp as a function of time for various template concentrations (error bars = s.d.; n = 3; R2 = 0.998). (f) The intercept (t0, exp) of the line in Fig. 5e and the threshold time Ct of real time, benchtop RT-LAMP curves as functions of the number of templates (error bars = s.d.; n = 3; R2 = 0.99). (g) XF, exp-XF,exp(3) as a function of the template number at various times t (error bars = s.d.; R2 = 0.99, n = 15). (h) The predicted position of the reaction front (XF, th) as a function of time for various numbers of templates. (i) The predicted intercept (t0, th) of the asymptotes in Fig. 5h as a function of template number.
Mentions: Fig. 5 analyzes the experimental data and compares it with the predictions of a simple theoretical model (to be described later). We take the emission intensity to be proportional to the amplicons' concentration c(x,t), assumed uniform in each cross-section of the conduit. Fig. 5a depicts as a function of position x at various times t. The lines and symbols correspond, respectively, to predictions and experimental data. We define the location of the reaction front XF(t) as the position at which . When x < XF, and the amplification reaction is nearly complete (the bright regions with fluorescent emission in Fig. 3). When x > XF, and no amplification has yet occurred (the dark regions in Fig. 3).

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