Furthermore, the feasibility of transferring the novel method to two other platforms, digital nucleic acid testing and electrochemical nucleic acid testing, is investigated.Ī novel method was successfully developed and named mediator displacement (MD) LAMP. The principal objectives of this thesis are first, to develop an improved sequence-specific detection method for LAMP with a simplified probe design, second, to verify its analytical performance and third, to validate the method by using it to analyze clinical samples. However, current state-of-the-art methods suffer from complex probe design and elaborate optimization work, which is required for the detection of different targets due to the use of target-specific fluorogenic probes. Special attention is given to the sequence-specific, fluorescence-based detection of LAMP, which allows the highly specific and simultaneous detection of various targets (multiplexing). Loop-mediated isothermal amplification (LAMP) stands out as a particularly robust and highly sensitive method. These are especially suitable for point-of-need (PON) applications. In the last 20 years, polymerase chain reaction (PCR) has been steadily replaced by isothermal alternatives. Nucleic acid amplification tests (NAATs) are an essential diagnostic tool throughout the field of life sciences, including clinical applications, food quality control, and environmental monitoring. In the future, real-time PCR master mixes could be augmented with up to five standardized fluorogenic URs, each emitting light at a different wavelength. The novel set of standardized URs we present here simplifies development of multiplex real-time PCR assays by requiring only the design of label-free probes. Analyses of MRSA DNA standards and DNA extracted from patient swab samples showed improved lower limits of detection (LoDs) by a factor of 2-5 when using quadruplex real-time MP PCR instead of HP PCR. Results were comparable to corresponding multiplex hydrolysis probe (HP) PCR, also designated as TaqMan PCR. Performance of the optimized URs was verified in multiplex real-time MP PCR for the detection of a pentaplex food panel and a quadruplex methicillin-resistant Staphylococcus aureus (MRSA) panel. To enable multiplex real-time MP PCR, we designed a set of five optimized URs with different fluorescent labels. Primers and probes can be shipped in solution.Mediator probe (MP) PCR is a real-time PCR approach that uses standardized universal fluorogenic reporter oligonucleotides (UR) in conjunction with label-free sequence-specific probes.They are HPLC-purified to remove impurities such as unconjugated dyes or truncated probes that can increase background signal and decrease assay sensitivity.Here's how Custom TaqMan probes set your assay up for success: The quality of your data is limited by the quality of the primers and probes you choose. Our TaqMan MGB (minor groove binder), TAMRA, and QSY custom probes deliver outstanding performance and are manufactured using the same facilities and raw materials as the TaqMan Assays that are featured in over 40,000 publications. Whether you want to simultaneously interrogate multiple targets, use your own bioinformatics to design a probe, or detect exotic targets that do not already have a predesigned assay, we offer the ideal probes to design your own assay. Choose from our collection of dual-labeled TaqMan probes and unlabeled oligos for use as qPCR primers.
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