Nanotips for single-step preparation of DNA for qPCR analysis

Abstract

PCR inhibitory substances in complex sample matrices can cause false negatives or underestimation of target concentration. This study assessed DNA heat treatment for reducing inhibition during qPCR analysis of human adenovirus (HAdV) in wastewater samples. Inhibition was reduced by heat treating DNA, where mean HAdV concentration was increased by 0.71 log 10 GC/L (and up to 3.04 log 10 GC/L in one case), and replicate variability and false negatives were reduced. DNA heat treatment should be further investigated for improving reliability of HAdV concentration estimates in water, which can support more accurate assessment of health risks associated with viral pathogen exposure.

Atomic Force Microscopic Detection Enabling Multiplexed Low-Cycle-Number Quantitative Polymerase Chain Reaction for Biomarker Assays

“…However, 0.1 μL or even less is required for deposition on the mica surface. Using advanced DNA purification techniques, such as purification by electric field, − we can potentially increase the sensitivity of this assay by 100×.…”

Nanostructured Tip-Shaped Biosensors: Application of Six Sigma Approach for Enhanced Manufacturing

“…Capillary action could enhance the sensitivity because the compressive pressure at the meniscus around a nanotip could result in the concentration and capture effects of target analyte in conjunction with viscosity [ 40 , 41 ]. Although the capture using capillary action is nonspecific, size selective sorting could be obtained on a nanotip surface because of the capillary force near the nanotip [ 40 , 42 ]. Using the selective sorting, a target could be collected in the mixture of different size particles through the withdrawal action of a nanotip.…”

“…Using the selective sorting, a target could be collected in the mixture of different size particles through the withdrawal action of a nanotip. The captured DNA using a nanotip could be amplified by real time polymerase chain reaction (qPCR) without further purification [ 42 ]. The nanotip composed of CNTs and SiC nanowires was dissolved in a PCR tube, which allowed for direct amplification of DNA in the reactor.…”

Recent Advances in Forensic DNA Analysis

“…Variations of the use of electric fields for DNA extraction include the use of nanotip concentrators made of SiC nanowires and single walled carbon nanotubes. DNA can be captured by nanotip concentrators in a single step and then be directly eluted into a PCR tube, reducing preparation time for further downstream analysis [89]. The use of a fourferrocene modified oligonuleotide has also been investigated as a probe for DNA detection and then used in a gold electrode microsystem.…”