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Multiple strategies to improve sensitivity, speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection.

Tong Y, Lemieux B, Kong H - BMC Biotechnol. (2011)

Bottom Line: The effect of combing all strategies was compared with that of the individual strategy.Some of them can be adjusted and applied to other formats of nucleic acid amplification.Furthermore, the strategies to improve the in vitro assays by maximally simulating the nature conditions may be useful in the general field of developing molecular assays.

View Article: PubMed Central - HTML - PubMed

Affiliation: BioHelix Corp, Beverly, MA, USA. tong@biohelix.com

ABSTRACT

Background: In the past decades the rapid growth of molecular diagnostics (based on either traditional PCR or isothermal amplification technologies) meet the demand for fast and accurate testing. Although isothermal amplification technologies have the advantages of low cost requirements for instruments, the further improvement on sensitivity, speed and robustness is a prerequisite for the applications in rapid pathogen detection, especially at point-of-care diagnostics. Here, we describe and explore several strategies to improve one of the isothermal technologies, helicase-dependent amplification (HDA).

Results: Multiple strategies were approached to improve the overall performance of the isothermal amplification: the restriction endonuclease-mediated DNA helicase homing, macromolecular crowding agents, and the optimization of reaction enzyme mix. The effect of combing all strategies was compared with that of the individual strategy. With all of above methods, we are able to detect 50 copies of Neisseria gonorrhoeae DNA in just 20 minutes of amplification using a nearly instrument-free detection platform (BESt™ cassette).

Conclusions: The strategies addressed in this proof-of-concept study are independent of expensive equipment, and are not limited to particular primers, targets or detection format. However, they make a large difference in assay performance. Some of them can be adjusted and applied to other formats of nucleic acid amplification. Furthermore, the strategies to improve the in vitro assays by maximally simulating the nature conditions may be useful in the general field of developing molecular assays. A new fast molecular assay for Neisseria gonorrhoeae has also been developed which has great potential to be used at point-of-care diagnostics.

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Related in: MedlinePlus

Effects of macromolecular crowding reagents. The effects are shown as the difference in detection time (calculated by: 2 × (Ttreaction without crowding agent- Ttreaction with crowding agent )) relative to non chemical control reactions versus crowding agents concentration in the final assay. The blue groups represent the effects of Ficoll, the green groups represent the effects of Dextran, the red groups represent the effects of PEG. The maximal tested concentration of each chemical is limited by its solubility (stock concentration) or viscosity (transferable or not by pipette).
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Figure 2: Effects of macromolecular crowding reagents. The effects are shown as the difference in detection time (calculated by: 2 × (Ttreaction without crowding agent- Ttreaction with crowding agent )) relative to non chemical control reactions versus crowding agents concentration in the final assay. The blue groups represent the effects of Ficoll, the green groups represent the effects of Dextran, the red groups represent the effects of PEG. The maximal tested concentration of each chemical is limited by its solubility (stock concentration) or viscosity (transferable or not by pipette).

Mentions: In order to examine the effect of macromolecular crowding on HDA, we tested the effect of numerous agents; i.e., polyethylene glycol (PEG) of 8 kilodaltons (8 K), PEG 20 K, PEG 35 K, Ficoll 70, Ficoll 400, Dextran 70 K, Dextran 500 K, Dextran 2000 K. The Tt values of amplifications with 50,000 copies of NG genomic DNA performed with different concentrations of these crowding agents were compared to the control reactions performed without the latter. As shown in Figure 2, HDA reactions performed under the conditions of macromolecular crowding were faster than the control reactions and the higher molecular weight crowding agents had the greatest effects on reaction speed. However, higher molecular weight chemicals also increased the viscosity of the solution, such that pipetting accuracy and mixing efficiency were affected. The upper limit of concentration of each chemical in HDA reaction was also limited by its solubility in stock solution. Although the PEG group had the most obvious effects, it also increased the incidence of primer dimer formation as measured by melt curve analysis (data not shown). Therefore, Ficoll and Dextran in final concentrations ranging from 5% to15% are better crowding agents for accelerating HDA.


Multiple strategies to improve sensitivity, speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection.

Tong Y, Lemieux B, Kong H - BMC Biotechnol. (2011)

Effects of macromolecular crowding reagents. The effects are shown as the difference in detection time (calculated by: 2 × (Ttreaction without crowding agent- Ttreaction with crowding agent )) relative to non chemical control reactions versus crowding agents concentration in the final assay. The blue groups represent the effects of Ficoll, the green groups represent the effects of Dextran, the red groups represent the effects of PEG. The maximal tested concentration of each chemical is limited by its solubility (stock concentration) or viscosity (transferable or not by pipette).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104364&req=5

Figure 2: Effects of macromolecular crowding reagents. The effects are shown as the difference in detection time (calculated by: 2 × (Ttreaction without crowding agent- Ttreaction with crowding agent )) relative to non chemical control reactions versus crowding agents concentration in the final assay. The blue groups represent the effects of Ficoll, the green groups represent the effects of Dextran, the red groups represent the effects of PEG. The maximal tested concentration of each chemical is limited by its solubility (stock concentration) or viscosity (transferable or not by pipette).
Mentions: In order to examine the effect of macromolecular crowding on HDA, we tested the effect of numerous agents; i.e., polyethylene glycol (PEG) of 8 kilodaltons (8 K), PEG 20 K, PEG 35 K, Ficoll 70, Ficoll 400, Dextran 70 K, Dextran 500 K, Dextran 2000 K. The Tt values of amplifications with 50,000 copies of NG genomic DNA performed with different concentrations of these crowding agents were compared to the control reactions performed without the latter. As shown in Figure 2, HDA reactions performed under the conditions of macromolecular crowding were faster than the control reactions and the higher molecular weight crowding agents had the greatest effects on reaction speed. However, higher molecular weight chemicals also increased the viscosity of the solution, such that pipetting accuracy and mixing efficiency were affected. The upper limit of concentration of each chemical in HDA reaction was also limited by its solubility in stock solution. Although the PEG group had the most obvious effects, it also increased the incidence of primer dimer formation as measured by melt curve analysis (data not shown). Therefore, Ficoll and Dextran in final concentrations ranging from 5% to15% are better crowding agents for accelerating HDA.

Bottom Line: The effect of combing all strategies was compared with that of the individual strategy.Some of them can be adjusted and applied to other formats of nucleic acid amplification.Furthermore, the strategies to improve the in vitro assays by maximally simulating the nature conditions may be useful in the general field of developing molecular assays.

View Article: PubMed Central - HTML - PubMed

Affiliation: BioHelix Corp, Beverly, MA, USA. tong@biohelix.com

ABSTRACT

Background: In the past decades the rapid growth of molecular diagnostics (based on either traditional PCR or isothermal amplification technologies) meet the demand for fast and accurate testing. Although isothermal amplification technologies have the advantages of low cost requirements for instruments, the further improvement on sensitivity, speed and robustness is a prerequisite for the applications in rapid pathogen detection, especially at point-of-care diagnostics. Here, we describe and explore several strategies to improve one of the isothermal technologies, helicase-dependent amplification (HDA).

Results: Multiple strategies were approached to improve the overall performance of the isothermal amplification: the restriction endonuclease-mediated DNA helicase homing, macromolecular crowding agents, and the optimization of reaction enzyme mix. The effect of combing all strategies was compared with that of the individual strategy. With all of above methods, we are able to detect 50 copies of Neisseria gonorrhoeae DNA in just 20 minutes of amplification using a nearly instrument-free detection platform (BESt™ cassette).

Conclusions: The strategies addressed in this proof-of-concept study are independent of expensive equipment, and are not limited to particular primers, targets or detection format. However, they make a large difference in assay performance. Some of them can be adjusted and applied to other formats of nucleic acid amplification. Furthermore, the strategies to improve the in vitro assays by maximally simulating the nature conditions may be useful in the general field of developing molecular assays. A new fast molecular assay for Neisseria gonorrhoeae has also been developed which has great potential to be used at point-of-care diagnostics.

Show MeSH
Related in: MedlinePlus