• Home
  • Search Results
  • A closed-tube detection of loop-mediated isothermal amplification (LAMP) products using a wax-sealed fluorescent intercalator.

A closed-tube detection of loop-mediated isothermal amplification (LAMP) products using a wax-sealed fluorescent intercalator.

Journal of nanoscience and nanotechnology (2013-07-19)
Chao Liang, Sijia Cheng, Yanan Chu, Haiping Wu, Binjie Zou, Huan Huang, Tao Xi, Guohua Zhou
ABSTRACT

LAMP is an isothermal amplification method that can achieve ultra-high sensitivity and specificity. However, the conventional detection of LAMP amplicons can lead to cross-contamination due to the need to open the reaction tube which contains a large number of amplicons. To achieve closed-tube LAMP detection, we have developed a method that separates a solution of SYBR Green I (SGI) from the LAMP reagents using temperature-sensitive wax. The SGI is sealed in the bottom of the tube so not to interfere with the LAMP reaction, but is released into the mixture after the completion of the reaction by melting the wax. To enable the analysis of the closed-tube LAMP samples automatically, an instrument based on this new method was constructed. The background measurement of the LAMP due to primer dimers was significantly reduced by detecting the amplicons at 75 degrees C. HBV and 2009 H1N1 virus were successfully analyzed by the LAMP assay using tubes containing wax-sealed SGI and the prototype instrument, indicating that the method has the advantage of easy set-up (no extra components need to be added into the LAMP mixture for detection), high sensitivity (fluorescent intercalator), low background (detected at 75 degrees C) and no cross-contamination (closed-tube). Therefore, the novel LAMP detection, coupled with the instrument has the potential to be a diagnostic tool for a number of clinical applications in hospitals as well as on-site screening of pathogenic agents.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Deoxyribonucleic acid, single stranded from salmon testes, For hybridization
Sigma-Aldrich
Deoxyribonucleic acid, single stranded from salmon testes, For hybridization
Sigma-Aldrich
Deoxyribonucleic acid sodium salt from herring testes, Type XIV
Sigma-Aldrich
Plasmid DNA from E. coli RRI, pUC19, buffered aqueous solution
Sigma-Aldrich
Deoxyribonucleic acid sodium salt from human placenta, Type XIII
Sigma-Aldrich
Deoxyribonucleic acid from human placenta, buffered aqueous solution, sexed, female
Sigma-Aldrich
Plasmid DNA from E. coli RRI, pUC18, buffered aqueous solution
Sigma-Aldrich
Deoxyribonucleic acid, single stranded from human placenta
Sigma-Aldrich
Deoxyribonucleic acid from Micrococcus luteus (lysodeikticus), Type XI, Highly polymerized