Rapid Detection of SARS-CoV-2 Variants of Concern by Genomic Surveillance Techniques.

This chapter describes the application of genomic, transcriptomic, proteomic, and metabolomic methods in the study of SARS-CoV-2 variants of concern. We also describe the important role of machine learning tools to identify the most significant biomarker signatures and discuss the latest point-of-care devices that can be used to translate these findings to the physician's office or to bedside care. The main emphasis is placed on increasing our diagnostic capacity and predictability of disease outcomes to guide the most appropriate treatment strategies.

Multiplex Quantitative Polymerase Chain Reaction Test to Identify SARS-CoV-2 Variants.

Quantitative polymerase chain reaction (qPCR) is a routinely used method for detection and quantitation of gene expression in real time. This is achieved through the incorporation and measurement of fluorescent reporter probes in the amplified cDNA strands, since the fluorescent signals increase as the reaction progresses. The availability of multiple probes which fluoresce at different wavelengths allows for multiplexing as this gives rise to amplicons with unique fluorescent signatures.

Multiplex Quantitative Polymerase Chain Reaction Diagnostic Test for SARS-CoV-2 and Influenza A/B Viruses.

COVID-19 disease caused by the novel SARS-CoV-2 virus represents a new challenge for healthcare systems. The molecular confirmation of infection is crucial to guide public health decision-making. This task could be made more difficult during the next influenza season.

Rapid and Easy Protocol for Quantification of Next-Generation Sequencing Libraries.

The emergence of next-generation sequencing (NGS) over the last 10 years has increased the efficiency of DNA sequencing in terms of speed, ease, and price. However, the exact quantification of a NGS library is crucial in order to obtain good data on sequencing platforms developed by the current market leader Illumina. Different approaches for DNA quantification are available currently and the most commonly used are based on analysis of the physical properties of the DNA through spectrophotometric or fluorometric methods.

Multiplex Single Nucleotide Polymorphism Analyses.

Quantitative polymerase chain reaction (qPCR) is a routinely used method for detection and quantitation of gene expression in real time. This is achieved through the incorporation and measurement of fluorescent reporter probes in the amplified cDNA strands, since the fluorescent signals increase as the reaction progresses. The availability of multiple probes that fluoresce at different wavelengths allows for multiplexing as this gives rise to amplicons with unique fluorescent signatures.

Multiplex Analysis Using cDNA Transcriptomic Profiling.

DNA microarrays contain microscopic DNA spots attached to a solid surface. Each spot contains picomolar levels of a specific DNA probe sequence and hybridization to the corresponding gene products can be detected and quantitated through the use of fluorescently labeled target DNA. In this format, DNA microarrays can be used to measure the expression level of thousands of genes in a single experiment.

Multiplex Analyses Using Real-Time Quantitative PCR.

Quantitative polymerase chain reaction (qPCR) is a routinely used method for the detection and quantitation of gene expression in real time. Multiplex qPCR requires the use of probe-based assays, in which each probe is labeled with a unique fluorescent dye, resulting in different observed colors for each assay. The signal from each dye is used to quantitate the amount of each target separately in the same tube or well.

Controlled elimination of clathrin heavy-chain expression in DT40 lymphocytes.

We exploited the high rate of homologous recombination shown by the chicken B cell line DT40 to inactivate the endogenous alleles for clathrin heavy chain and replace them with human clathrin complementary DNA under the control of a tetracycline-regulatable promoter. Clathrin repression perturbed the activities of Akt-mediated and mitogen-activated protein kinase-mediated signaling pathways and induced apoptosis; this finding suggests that in DT40 cells clathrin helps to maintain the integrity of antiapoptotic survival pathways. We also describe a variant cell line in which these signaling pathways were unaffected by clathrin down-regulation.