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Loop-Mediated Amplification Accelerated by Stem Primers
Lumora Ltd., Unit 4, Cambridgeshire Business Park, Bartholomews Walk, Ely, Cambridgeshire CB7 4EA, UK
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Received: 31 August 2011; in revised form: 6 November 2011 / Accepted: 29 November 2011 / Published: 8 December 2011
Abstract: Isothermal nucleic acid amplifications (iNAATs) have become an important alternative to PCR for in vitro molecular diagnostics in all fields. Amongst iNAATs Loop-mediated amplification (LAMP) has gained much attention over the last decade because of the simplicity of hardware requirements. LAMP demonstrates performance equivalent to that of PCR, but its application has been limited by the challenging primer design. The design of six primers in LAMP requires a selection of eight priming sites with significant restrictions imposed on their respective positioning and orientation. In order to relieve primer design constraints we propose an alternative approach which uses Stem primers instead of Loop primers and demonstrate the application of STEM-LAMP in assaying for Clostridium difficile, Listeria monocytogenes and HIV. Stem primers used in LAMP in combination with loop-generating and displacement primers gave significant benefits in speed and sensitivity, similar to those offered by Loop primers, while offering additional options of forward and reverse orientations, multiplexing, use in conjunction with Loop primers or even omission of one or two displacement primers, where necessary. Stem primers represent a valuable alternative to Loop primers and an additional tool for IVD assay development by offering more choices for primer design at the same time increasing assay speed, sensitivity, and reproducibility.
Keywords: stem primers; isothermal amplification; loop-mediated amplification; in vitro diagnostics; Clostridium difficile; Listeria monocytogenes; HIV
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Gandelman, O.; Jackson, R.; Kiddle, G.; Tisi, L. Loop-Mediated Amplification Accelerated by Stem Primers. Int. J. Mol. Sci. 2011, 12, 9108-9124.
Gandelman O, Jackson R, Kiddle G, Tisi L. Loop-Mediated Amplification Accelerated by Stem Primers. International Journal of Molecular Sciences. 2011; 12(12):9108-9124.
Gandelman, Olga; Jackson, Rebecca; Kiddle, Guy; Tisi, Laurence. 2011. "Loop-Mediated Amplification Accelerated by Stem Primers." Int. J. Mol. Sci. 12, no. 12: 9108-9124.