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

Schematic diagram of lateral flow test strip. (a) a typical structure of the lateral flow test strip; (b) Seen on the membrane are the “test line” and “control line”. The test line quantifies the target in the sample while the control line confirms adequate hybridization conditions. The capture and control probes are immobilized on the membrane prior to the assay. HRP indicates the horseradish peroxidase and StAv indicates streptavidin; (c) conjugation between HRP and nucleic acid via the interaction between biotin and streptavidin.
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biosensors-02-00032-f001: Schematic diagram of lateral flow test strip. (a) a typical structure of the lateral flow test strip; (b) Seen on the membrane are the “test line” and “control line”. The test line quantifies the target in the sample while the control line confirms adequate hybridization conditions. The capture and control probes are immobilized on the membrane prior to the assay. HRP indicates the horseradish peroxidase and StAv indicates streptavidin; (c) conjugation between HRP and nucleic acid via the interaction between biotin and streptavidin.

Mentions: The test strip consists of four components mounted together on an adhesive backing: sample application pad, oligonucleotide-biotin-streptavidin-HRP conjugate release pad, nitrocellulose membrane, and the absorbent pad (Figure 1). Each component was prepared separately and then assembled on an adhesive backing prior to use. Once assembled, the strips were stored desiccated at 4 °C until use.


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

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

Schematic diagram of lateral flow test strip. (a) a typical structure of the lateral flow test strip; (b) Seen on the membrane are the “test line” and “control line”. The test line quantifies the target in the sample while the control line confirms adequate hybridization conditions. The capture and control probes are immobilized on the membrane prior to the assay. HRP indicates the horseradish peroxidase and StAv indicates streptavidin; (c) conjugation between HRP and nucleic acid via the interaction between biotin and streptavidin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

biosensors-02-00032-f001: Schematic diagram of lateral flow test strip. (a) a typical structure of the lateral flow test strip; (b) Seen on the membrane are the “test line” and “control line”. The test line quantifies the target in the sample while the control line confirms adequate hybridization conditions. The capture and control probes are immobilized on the membrane prior to the assay. HRP indicates the horseradish peroxidase and StAv indicates streptavidin; (c) conjugation between HRP and nucleic acid via the interaction between biotin and streptavidin.
Mentions: The test strip consists of four components mounted together on an adhesive backing: sample application pad, oligonucleotide-biotin-streptavidin-HRP conjugate release pad, nitrocellulose membrane, and the absorbent pad (Figure 1). Each component was prepared separately and then assembled on an adhesive backing prior to use. Once assembled, the strips were stored desiccated at 4 °C until use.

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