Rapid visual identification of PCR amplified nucleic acids by centrifugal gel separation: Potential use for molecular point-of-care tests

Biosensors & Bioelectronics
Sang-Hyun HwangSang Jun Son

Abstract

Recently, nucleic acid amplification and detection techniques have progressed based on advances in in microfluidics, microelectronics, and optical systems. Nucleic acids amplification based point-of-care test (POCT) in resource-limited settings requires simple visual detection methods. Several biosensing methods including lateral flow immunoassays (LFIA) were previously used to visually detect nucleic acids. However, prolonged assay time, several washing steps, and a need for specific antibodies limited their use. Here we developed a novel, rapid method to visualize amplified nucleic acids with naked eyes in clinical samples. First, we optimized conditions based on separation using very low centrifugal force and a density medium to detect human papillomavirus (HPV)-16 DNA in cervical specimens. After DNA extraction, HPV16 PCR was performed with biotin-labeled forward primer and Cy3-labeled reverse primer. PCR amplicon was mixed with streptavidin-magnetic beads, introduced into the density medium. After two-minute centrifugation, the result was visually identified. This system showed identical results with commercial HPV real-time PCR for 30 clinical samples and could detect up to 10(2)copies/mL of HPV DNA without any optical in...Continue Reading

References

Apr 11, 1995·Nucleic Acids Research·H S Rye, A N Glazer
Aug 1, 1993·Transfusion·K J ReisT M Setcavage
Dec 18, 2013·The Lancet Infectious Diseases·Paul K DrainIngrid V Bassett
Mar 29, 2014·Electrophoresis·Wendell Karlos Tomazelli ColtroDosil Pereira de Jesus

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