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A novel thermostable polymerase for RNA and DNA loop-mediated isothermal amplification (LAMP).

Chander Y, Koelbl J, Puckett J, Moser MJ, Klingele AJ, Liles MR, Carrias A, Mead DA, Schoenfeld TW - Front Microbiol (2014)

Bottom Line: Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst) and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions.Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates.Results of this study show that OmniAmp Pol is ideally suited for low resource molecular detection of pathogens.

View Article: PubMed Central - PubMed

Affiliation: Lucigen Corporation Middleton, WI, USA.

ABSTRACT
Meeting the goal of providing point of care (POC) tests for molecular detection of pathogens in low resource settings places stringent demands on all aspects of the technology. OmniAmp DNA polymerase (Pol) is a thermostable viral enzyme that enables true POC use in clinics or in the field by overcoming important barriers to isothermal amplification. In this paper, we describe the multiple advantages of OmniAmp Pol as an isothermal amplification enzyme and provide examples of its use in loop-mediated isothermal amplification (LAMP) for pathogen detection. The inherent reverse transcriptase activity of OmniAmp Pol allows single enzyme detection of RNA targets in RT-LAMP. Common methods of nucleic acid amplification are highly susceptible to sample contaminants, necessitating elaborate nucleic acid purification protocols that are incompatible with POC or field use. OmniAmp Pol was found to be less inhibited by whole blood components typical in certain crude sample preparations. Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst) and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions. Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates. Molecular diagnostics in field settings can be challenging due to the lack of refrigeration. The stability of OmniAmp Pol is compatible with a dry format that enables long term storage at ambient temperatures. A final requirement for field operability is compatibility with either commonly available instruments or, in other cases, a simple, inexpensive, portable detection mode requiring minimal training or power. Detection of amplification products is shown using lateral flow strips and analysis on a real-time PCR instrument. Results of this study show that OmniAmp Pol is ideally suited for low resource molecular detection of pathogens.

No MeSH data available.


(A) Beads of dried LAMP reagents in PCR tubes. (B) Feasibility data showing stability based on TTR of LAMP reagents in dried format stored at the indicated temperatures compared to wet enzyme stored at −20° over 180 days.
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Figure 9: (A) Beads of dried LAMP reagents in PCR tubes. (B) Feasibility data showing stability based on TTR of LAMP reagents in dried format stored at the indicated temperatures compared to wet enzyme stored at −20° over 180 days.

Mentions: A lyophilized formulation for the LAMP reagents was compared with wet reagents for detection of MS2 RNA phage target. No diminution of TTR or sensitivity was seen with the dried formulation compared to wet (Figure 8A) nor was an increase in non-specific amplification seen by visualization of LAMP products on a 2% agarose gel (Figure 8B). To evaluate stability, the lyophilized reaction mix (Figure 9A) was incubated at 23, 37, and 45°C and assayed in a Clostridium difficile LAMP reaction compared to wet reagent stored at −20°C (Figure 9B). The dried LAMP formulation was stable at 23°C and 37°C for 180 days. The dried reagent was stable at 45°C for 50 days although it did show a measurable drop in TTR after 90 days.


A novel thermostable polymerase for RNA and DNA loop-mediated isothermal amplification (LAMP).

Chander Y, Koelbl J, Puckett J, Moser MJ, Klingele AJ, Liles MR, Carrias A, Mead DA, Schoenfeld TW - Front Microbiol (2014)

(A) Beads of dried LAMP reagents in PCR tubes. (B) Feasibility data showing stability based on TTR of LAMP reagents in dried format stored at the indicated temperatures compared to wet enzyme stored at −20° over 180 days.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117986&req=5

Figure 9: (A) Beads of dried LAMP reagents in PCR tubes. (B) Feasibility data showing stability based on TTR of LAMP reagents in dried format stored at the indicated temperatures compared to wet enzyme stored at −20° over 180 days.
Mentions: A lyophilized formulation for the LAMP reagents was compared with wet reagents for detection of MS2 RNA phage target. No diminution of TTR or sensitivity was seen with the dried formulation compared to wet (Figure 8A) nor was an increase in non-specific amplification seen by visualization of LAMP products on a 2% agarose gel (Figure 8B). To evaluate stability, the lyophilized reaction mix (Figure 9A) was incubated at 23, 37, and 45°C and assayed in a Clostridium difficile LAMP reaction compared to wet reagent stored at −20°C (Figure 9B). The dried LAMP formulation was stable at 23°C and 37°C for 180 days. The dried reagent was stable at 45°C for 50 days although it did show a measurable drop in TTR after 90 days.

Bottom Line: Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst) and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions.Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates.Results of this study show that OmniAmp Pol is ideally suited for low resource molecular detection of pathogens.

View Article: PubMed Central - PubMed

Affiliation: Lucigen Corporation Middleton, WI, USA.

ABSTRACT
Meeting the goal of providing point of care (POC) tests for molecular detection of pathogens in low resource settings places stringent demands on all aspects of the technology. OmniAmp DNA polymerase (Pol) is a thermostable viral enzyme that enables true POC use in clinics or in the field by overcoming important barriers to isothermal amplification. In this paper, we describe the multiple advantages of OmniAmp Pol as an isothermal amplification enzyme and provide examples of its use in loop-mediated isothermal amplification (LAMP) for pathogen detection. The inherent reverse transcriptase activity of OmniAmp Pol allows single enzyme detection of RNA targets in RT-LAMP. Common methods of nucleic acid amplification are highly susceptible to sample contaminants, necessitating elaborate nucleic acid purification protocols that are incompatible with POC or field use. OmniAmp Pol was found to be less inhibited by whole blood components typical in certain crude sample preparations. Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst) and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions. Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates. Molecular diagnostics in field settings can be challenging due to the lack of refrigeration. The stability of OmniAmp Pol is compatible with a dry format that enables long term storage at ambient temperatures. A final requirement for field operability is compatibility with either commonly available instruments or, in other cases, a simple, inexpensive, portable detection mode requiring minimal training or power. Detection of amplification products is shown using lateral flow strips and analysis on a real-time PCR instrument. Results of this study show that OmniAmp Pol is ideally suited for low resource molecular detection of pathogens.

No MeSH data available.