Real-Time Duplex Applications of Loop-Mediated AMPlification (LAMP) by Assimilating Probes

International Journal of Molecular Sciences

Ryo Kubota²  Daniel M. Jenkins¹ 

[1] Department of Molecular Biosciences and Bioengineering, University of Hawai'.i, Mānoa, 1955 East-West Road, Honolulu, HI 96822, USA; E-Mail:;Diagenetix, Inc., Honolulu, HI 96822, USA

关键词: molecular diagnostics;    isothermal nucleic acid amplification;    Ralstonia solanacearum;    Race 3 Biovar 2;    Salmonella enterica;    point-of-care testing;    on-site detection;    on-site diagnostics;   

DOI  :  10.3390/ijms16034786

来源: mdpi

【 摘 要 】

Isothermal nucleic-acid amplification methods such as Loop-Mediated isothermal AMPlification (LAMP) are increasingly appealing alternatives to PCR for use in portable diagnostic system due to the low cost, weight, and power requirements of the instrumentation. As such, interest in developing new probes and other functionality based on the LAMP reaction has been intense. Here, we report on the development of duplexed LAMP assays for pathogen detection using spectrally unique Assimilating Probes. As proof of principle, we used a reaction for Salmonella enterica as a model coupled with a reaction for λ-phage DNA as an internal control, as well as a duplexed assay to sub-type specific quarantine strains of the bacterial wilt pathogen Ralstonia solanacearum. Detection limits for bacterial DNA analyzed in individual reactions was less than 100 genomic equivalents in all cases, and increased by one to two orders of magnitude when reactions were coupled in duplexed formats. Even so, due to the more robust activity of newly available strand-displacing polymerases, the duplexed assays reported here were more powerful than analogous individual reactions reported only a few years ago, and represent a significant advance for incorporation of internal controls to validate assay results in the field.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.