4.7 Article

One-pot isothermal DNA amplification - Hybridisation and detection by a disc-based method

Journal

SENSORS AND ACTUATORS B-CHEMICAL
Volume 204, Issue -, Pages 273-281

Publisher

ELSEVIER SCIENCE SA
DOI: 10.1016/j.snb.2014.07.073

Keywords

Isothermal solid-phase amplification; Compact disc; Pathogen; Microarray

Funding

  1. Project (Generalitat Valenciana) [GVA-PROMETEO/2010/008]
  2. Project (MINECO) [CTQ/2013/45875-R]
  3. Spanish Ministry of Education and Science

Ask authors/readers for more resources

An integrated sensor comprising isothermal DNA amplification and in situ detection is presented. The method principle is based on recombinase polymerase amplification (RPA) and detection in the microarray format by compact disc technology as a high-throughput sensing platform. Primers were immobilised on the polycarbonate surface of digital versatile discs (DVD) and, after hemi-nested amplification, multiplexing identification of each tethered product was achieved by optical scanning with a 650 nm-laser of the DVD drive. The efficiency of one-pot hybridisation/elongation/detection depended strongly on probedensity and other factors such as the concentration of the unbound primers present in solution. The optimised conditions provided equivalent amplification factors (7.3 x 10(8) -8.9 x 10(8) fold) to those obtained by conventional reactions performed in vials. The proposed method was applied to Salmonella detection (generic by hns and oriC genes, and specific for subspecies I by STM4507 gene). A triplex assay was satisfactorily compared to the non-integrated protocols. Food and vaccine samples were analysed in a shorter time with less handling. The results indicate that the multiplex DVD assay is a simple, competitive, isothermal, portable system that is particularly useful for microbiological routine analysis. (C) 2014 Elsevier B.V. All rights reserved.

Authors

I am an author on this paper
Click your name to claim this paper and add it to your profile.

Reviews

Primary Rating

4.7
Not enough ratings

Secondary Ratings

Novelty
-
Significance
-
Scientific rigor
-
Rate this paper

Recommended

No Data Available
No Data Available