Multiple strategies to improve sensitivity, speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection
- نوع فایل : کتاب
- زبان : انگلیسی
- مؤلف : Yanhong Tong (tong@biohelix.com) Bertrand Lemieux (lemieux@biohelix.com) Huimin Kong (kong@biohelix.com)
- چاپ و سال / کشور: 2011
Description
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, helicasedependent 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 (BEStTM cassette). Conclusions The strategies addressed in this proof-of-concept study are independent of expensive equipments, 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
BMC Biotechnology 2011, 11:50 doi:10.1186/1472-6750-11-50 ISSN 1472-6750 Article type Methodology article Submission date 4 January 2011 Acceptance date 11 May 2011 Publication date 11 May 2011