Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification

Molecules

De-Guo Wang¹  Jeffrey D. Brewster²  Moushumi Paul²  Peggy M. Tomasula² 

[1] Henan Postdoctoral Research Base, Food and Bioengineering College, Xuchang University, Xuchang 461000, China;Eastern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Wyndmoor, PA 19038, USA; E-Mails:

关键词: loop-mediated isothermal amplification (LAMP);    non-specific amplification;    dimethyl sulfoxide (DMSO);    Touchdown LAMP;   

DOI  :  10.3390/molecules20046048

来源: mdpi

【 摘 要 】

The technique of loop-mediated isothermal amplification (LAMP) utilizes four (or six) primers targeting six (or eight) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an increased likelihood of non-specific amplification induced by primer dimers. In this study, a set of LAMP primers were designed targeting the prfA gene sequence of Listeria monocytogenes, and dimethyl sulfoxide (DMSO) as well as Touchdown LAMP were employed to increase the sensitivity and specificity of the LAMP reactions. The results indicate that the detection limit of this novel LAMP assay with the newly designed primers and additives was 10 fg per reaction, which is ten-fold more sensitive than a commercial Isothermal Amplification Kit and hundred-fold more sensitive than previously reported LAMP assays. This highly sensitive LAMP assay has been shown to detect 11 strains of Listeria monocytogenes, and does not detect other Listeria species (including Listeria innocua and Listeria invanovii), providing some advantages in specificity over commercial Isothermal Amplification Kits and previously reported LAMP assay.

【 授权许可】

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