An improved and readily available version of Bst DNA Polymerase for LAMP, and applications to COVID-19 diagnostics
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
Despite the fact that strand-displacing activity is of great utility for a variety of applications, including isothermal amplification assays, there are relatively few strand-displacing DNA polymerases. In particular, the thermotolerant DNA polymerase from Geobacillus stearothermophilus ( previously Bacillus stearothermophilus), Bst DNA polymerase ( Bst DNAP), is used in a variety of assays, including loop-mediated isothermal amplification. However, despite its wide use, its properties remain open to improvement, as has been demonstrated by a variety of engineering efforts, including the identification of point mutations that impact its robustness, strand-displacement capabilities, and nascent reverse transcriptase activity.
Interestingly, a strategy that has been commonly used to alter the capabilities of DNA polymerases, the addition of additional DNA- or RNA-binding domains, has yet to be applied to Bst DNAP. To this end, we now show that by adding fusion domains the performance of Bst DNAP in isothermal amplification assays, including its nascent RT activity, can be greatly improved. The impact of these improvements on the development of LAMP assays for the detection of SARS-CoV-2 is fully explored.
Article activity feed
-
SciScore for 10.1101/2020.10.02.20203356: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources All assays were incubated in a thermocycler maintained at 65 °C for 60 min following which endpoint OSD fluorescence was imaged using a ChemiDoc camera (Bio-Rad Laboratories, Hercules, CA, USA). Bio-Rad Laboratoriessuggested: (Bio-Rad Laboratories, RRID:SCR_008426)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section …SciScore for 10.1101/2020.10.02.20203356: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources All assays were incubated in a thermocycler maintained at 65 °C for 60 min following which endpoint OSD fluorescence was imaged using a ChemiDoc camera (Bio-Rad Laboratories, Hercules, CA, USA). Bio-Rad Laboratoriessuggested: (Bio-Rad Laboratories, RRID:SCR_008426)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a protocol registration statement.
-
