Loop-mediated isothermal amplification in disposable polyester-toner microdevices

Author(s)

de Oliveira, Kezia Gomes ; Borba, Juliane Cristina ; Bailao, Alexandre Melo ; de Almeida Soares, Celia Maria ; Carrilho, Emanuel ; Mendes Duarte, Gabriela Rodrigues

• Document type: Journal article
• Source: Analytical Biochemistry; v. 534, p. 70-77, OCT 1 2017.
• Web of Science Citations: 3

Abstract

In recent years, isothermal DNA amplification methods have emerged as an alternative in molecular diagnostics due to its ease of operation. The purpose of using isothermal amplification is to simplify the diagnostics work by i) eliminating heating cycles, ii) reducing equipment costs, and iii) providing rapid and accurate results in laboratories with limited resources. Here we show a simple and fast method for E. coil detection in disposable polyester-toner (PeT) microdevice. The amplification by LAMP of the malB gene from E. coli was carried out in a microchamber with 5-mu L capacity and the reaction was thermally controlled with a thermoblock at 66 degrees C for 60 min. The passivation of the surface of PeT channels with BSA improved the efficiency of the LAMP reaction. The detection of amplified LAMP fragments was performed directly on-chip by visual detection and validated with off-chip detection to compare results. Visualization of amplicons directly in the microchip yielded positive reactions as low as 10 double-stranded DNA copies. Separation by gel electrophoresis was able to detect amplicons in reactions that initiated only with one copy of double-stranded DNA. We demonstrate that LAMP in PeT microchip is an important tool for molecular diagnostics at the point-of-care. (C) 2017 Published by Elsevier Inc. (AU)

• FAPESP's process: 15/17145-7 - A fully integrated polyester-toner (PT) based microchip for genetic analyses: Development and applications
• Grantee: Emanuel Carrilho
• Support Opportunities: Regular Research Grants