Limits...
Development of chemiluminescent lateral flow assay for the detection of nucleic acids.

Wang Y, Fill C, Nugen SR - Biosensors (Basel) (2012)

Bottom Line: Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety.On-membrane enzymatic signal amplification is used to reduce the limit of detection to the sub-femtomol level.The limit of detection was determined to be 0.5 fmols of nucleic acid target.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science, University of Massachusetts, 102 Holdsworth Way, Amherst, MA 01003, USA. yuhongw@foodsci.umass.edu.

ABSTRACT
Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety. Herein we report the development of a nucleic acid sequence-based lateral flow assay which achieves a low limit of detection using chemiluminescence. On-membrane enzymatic signal amplification is used to reduce the limit of detection to the sub-femtomol level. To demonstrate this assay, we detected synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, with relevance in human and animal health in sub-Saharan Africa. The intensity of the chemiluminescent signal was evaluated by using a charge-coupled device as well as a microtiter plate reader. We demonstrated that our lateral flow chemiluminescent assay has a very low limit of detection and is easy to use. The limit of detection was determined to be 0.5 fmols of nucleic acid target.

No MeSH data available.


Related in: MedlinePlus

Dose response for increasing nucleic acid target. Signal area was calculated by integrating the test lines from the microtiter plate reader (Figure 4). The logarithmic model was fitted to the non-zero values tested. Data points represent an average of a minimum of three replicates and error bars represent the standard deviation. The grey data point at target concentration of zero represents the background signal and was not used in the fitting of the logarithmic model.
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biosensors-02-00032-f005: Dose response for increasing nucleic acid target. Signal area was calculated by integrating the test lines from the microtiter plate reader (Figure 4). The logarithmic model was fitted to the non-zero values tested. Data points represent an average of a minimum of three replicates and error bars represent the standard deviation. The grey data point at target concentration of zero represents the background signal and was not used in the fitting of the logarithmic model.

Mentions: A logarithmic model was fitted to the dose response curve as seen in Figure 5 (R2 = 0.998). The limit of detection was determined using the 0 + 3SD method [28,29,30]. In this case, the background signal (i.e., signal produced when the concentration of target analyte is zero) plus 3 times the SD at background are used as the minimum detectable sample. The corresponding target level was then determined using the logarithmic model. A limit of detection of approximately 0.5 fmol was determined by this 0 + 3SD method. A general linear model ANOVA was performed on the experimental data obtained at both background (0.0 fmol target analyte) and limit of detection (0.5 fmol target analyte) dose response data and statistical significance was determined at P < 0.01.


Development of chemiluminescent lateral flow assay for the detection of nucleic acids.

Wang Y, Fill C, Nugen SR - Biosensors (Basel) (2012)

Dose response for increasing nucleic acid target. Signal area was calculated by integrating the test lines from the microtiter plate reader (Figure 4). The logarithmic model was fitted to the non-zero values tested. Data points represent an average of a minimum of three replicates and error bars represent the standard deviation. The grey data point at target concentration of zero represents the background signal and was not used in the fitting of the logarithmic model.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263543&req=5

biosensors-02-00032-f005: Dose response for increasing nucleic acid target. Signal area was calculated by integrating the test lines from the microtiter plate reader (Figure 4). The logarithmic model was fitted to the non-zero values tested. Data points represent an average of a minimum of three replicates and error bars represent the standard deviation. The grey data point at target concentration of zero represents the background signal and was not used in the fitting of the logarithmic model.
Mentions: A logarithmic model was fitted to the dose response curve as seen in Figure 5 (R2 = 0.998). The limit of detection was determined using the 0 + 3SD method [28,29,30]. In this case, the background signal (i.e., signal produced when the concentration of target analyte is zero) plus 3 times the SD at background are used as the minimum detectable sample. The corresponding target level was then determined using the logarithmic model. A limit of detection of approximately 0.5 fmol was determined by this 0 + 3SD method. A general linear model ANOVA was performed on the experimental data obtained at both background (0.0 fmol target analyte) and limit of detection (0.5 fmol target analyte) dose response data and statistical significance was determined at P < 0.01.

Bottom Line: Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety.On-membrane enzymatic signal amplification is used to reduce the limit of detection to the sub-femtomol level.The limit of detection was determined to be 0.5 fmols of nucleic acid target.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science, University of Massachusetts, 102 Holdsworth Way, Amherst, MA 01003, USA. yuhongw@foodsci.umass.edu.

ABSTRACT
Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety. Herein we report the development of a nucleic acid sequence-based lateral flow assay which achieves a low limit of detection using chemiluminescence. On-membrane enzymatic signal amplification is used to reduce the limit of detection to the sub-femtomol level. To demonstrate this assay, we detected synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, with relevance in human and animal health in sub-Saharan Africa. The intensity of the chemiluminescent signal was evaluated by using a charge-coupled device as well as a microtiter plate reader. We demonstrated that our lateral flow chemiluminescent assay has a very low limit of detection and is easy to use. The limit of detection was determined to be 0.5 fmols of nucleic acid target.

No MeSH data available.


Related in: MedlinePlus