Development of an Automated, Non-Enzymatic Nucleic Acid Amplification Test

Micromachines

Nicholas M. Adams¹  Hannah E. Stepp¹  Zackary A. Zimmers¹  Alexander D. Boyd¹  Frederick R. Haselton¹ 

[1] Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37212, USA;

关键词: automation;    non-enzymatic;    DNA amplification;    L-DNA;    microfluidic;    fluorescence;   

DOI  :  10.3390/mi12101204

来源: DOAJ

【 摘 要 】

Among nucleic acid diagnostic strategies, non-enzymatic tests are the most promising for application at the point of care in low-resource settings. They remain relatively under-utilized, however, due to inadequate sensitivity. Inspired by a recent demonstration of a highly-sensitive dumbbell DNA amplification strategy, we developed an automated, self-contained assay for detection of target DNA. In this new diagnostic platform, called the automated Pi-powered looping oligonucleotide transporter, magnetic beads capture the target DNA and are then loaded into a microfluidic reaction cassette along with the other reaction solutions. A stepper motor controls the motion of the cassette relative to an external magnetic field, which moves the magnetic beads through the reaction solutions automatically. Real-time fluorescence is used to measure the accumulation of dumbbells on the magnetic bead surface. Left-handed DNA dumbbells produce a distinct signal which reflects the level of non-specific amplification, acting as an internal control. The autoPiLOT assay detected as little as 5 fM target DNA, and was also successfully applied to the detection of S. mansoni DNA. The autoPiLOT design is a novel step forward in the development of a sensitive, user-friendly, low-resource, non-enzymatic diagnostic test.

【 授权许可】

Unknown