Dataset Information

Passivated Impedimetric Sensors for Immobilization-Free Pathogen Detection by Isothermal Amplification and Melt Curve Analysis.

ABSTRACT: The ongoing SARS-CoV-2 pandemic demonstrates that the capacity of centralized clinical diagnosis laboratories represents a significant limiting factor in the global fight against the newly emerged virus. Scaling up these capacities also requires simple and robust methods for virus diagnosis to be easily driven by untrained personnel in a point-of-care (POC) environment. The use of impedance sensors reduces the complexity and costs of diagnostic instruments and increases automation of diagnosis processes. We present an impedance point-of-care system (IMP-POCS) that uses interdigitated electrodes surrounded by an integrated heating meander to monitor loop-mediated isothermal amplification (LAMP) and melt curve analysis (MCA) consecutively in a short time. MCA permits distinguishing false- from true-positive results and significantly raises the validity of pathogen detection. Conclusively, the herein-developed miniaturized total analysis system (µTAS) represents a powerful and promising tool for providing reliable, low-cost alternatives to standard clinical diagnosis.

SUBMITTER: Steinmaßl M

PROVIDER: S-EPMC9138539 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Passivated Impedimetric Sensors for Immobilization-Free Pathogen Detection by Isothermal Amplification and Melt Curve Analysis.

Steinmaßl Matthias M Boudaden Jamila J Edgü Güven G Freund Lena Julie LJ Meyer Simon S Mordehay Noa N Soto Melissa M Endres Hanns-Erik HE Muth Jost J Prüfer Dirk D Lerch Wilfried W Kutter Christoph C

Biosensors 20220420 5

The ongoing SARS-CoV-2 pandemic demonstrates that the capacity of centralized clinical diagnosis laboratories represents a significant limiting factor in the global fight against the newly emerged virus. Scaling up these capacities also requires simple and robust methods for virus diagnosis to be easily driven by untrained personnel in a point-of-care (POC) environment. The use of impedance sensors reduces the complexity and costs of diagnostic instruments and increases automation of diagnosis p ...[more]

PMID: 35624562

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Project description:BackgroundMethylation of DNA is a common mechanism for silencing genes, and aberrant methylation is increasingly being implicated in many diseases such as cancer. There is a need for robust, inexpensive methods to quantitate methylation across a region containing a number of CpGs. We describe and validate a rapid, in-tube method to quantitate DNA methylation using the melt data obtained following amplification of bisulfite modified DNA in a real-time thermocycler.MethodsWe first describe a mathematical method to normalise the raw fluorescence data generated by heating the amplified bisulfite modified DNA. From this normalised data the temperatures at which melting begins and finishes can be calculated, which reflect the less and more methylated template molecules present respectively. Also the T50, the temperature at which half the amplicons are melted, which represents the summative methylation of all the CpGs in the template mixture, can be calculated. These parameters describe the methylation characteristics of the region amplified in the original sample.ResultsFor validation we used synthesized oligonucleotides and DNA from fresh cells and formalin fixed paraffin embedded tissue, each with known methylation. Using our quantitation we could distinguish between unmethylated, partially methylated and fully methylated oligonucleotides mixed in varying ratios. There was a linear relationship between T50 and the dilution of methylated into unmethylated DNA. We could quantitate the change in methylation over time in cell lines treated with the demethylating drug 5-aza-2'-deoxycytidine, and the differences in methylation associated with complete, clonal or no loss of MGMT expression in formalin fixed paraffin embedded tissues.ConclusionWe have validated a rapid, simple in-tube method to quantify methylation which is robust and reproducible, utilizes easily designed primers and does not need proprietary algorithms or software. The technique does not depend on any operator manipulation or interpretation of the melt curves, and is suitable for use in any laboratory with a real-time thermocycler. The parameters derived provide an objective description and quantitation of the methylation in a specimen, and can be used to for statistical comparisons of methylation between specimens.

Dataset Information

Passivated Impedimetric Sensors for Immobilization-Free Pathogen Detection by Isothermal Amplification and Melt Curve Analysis.

Publications

Passivated Impedimetric Sensors for Immobilization-Free Pathogen Detection by Isothermal Amplification and Melt Curve Analysis.

Similar Datasets

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