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An improved nucleic acid sequence-based amplification method mediated by T4 gene 32 protein
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posted on 2022-03-24, 00:00 authored by Y H Nai, Egan DoevenEgan Doeven, Rosanne GuijtRosanne GuijtThe uptake of Nucleic Acid Sequence-Based Amplification (NASBA) for point of care testing may be hindered by a complexity in the workflow due the requirement of a thermal denaturation step to initiate the cyclic isothermal amplification before the addition of the amplification enzymes. Despite reports of successful enhancement of other DNA and RNA amplification methods using DNA and RNA binding proteins, this has not been reported for NASBA. Here, three single-stranded binding proteins, RecA, Extreme Thermostable Single-stranded binding protein (ET SSB) and T4 gene gp32 protein (gp32), were incorporated in NASBA protocol and used for single pot, one-step NASBA at 41 °C. Indeed, all SSBs showed significantly improved amplifications compared with the 2-step process, but only gp32 showed no non-specific aberrant amplification, and slightly improved the time-to-positivity in comparison with the conventional NASBA. For synthetic HIV-1 RNA, gp32 was found to improve the time-to-positivity (ttp) by average of 13.6% of one-step NASBA and 6.7% of conventional NASBA for the detection of HIV-1 RNA, showing its potential for simplifying the workflow as desirable for point of care applications of NASBA.
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Journal
PLoS ONEVolume
17Issue
3Article number
ARTN e0265391Pagination
1 - 9Publisher
Public Library of Science (PLoS)Location
San Francisco, Calif.Publisher DOI
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1932-6203eISSN
1932-6203Language
EnglishPublication classification
C1 Refereed article in a scholarly journalUsage metrics
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