Infectious Diseases Testing

Abstract

Molecular methods have been applied widely for the diagnosis of infectious diseases. Beginning with solution hybridization in the early 1990s, multiple methods for nucleic acid hybridization and amplification have been introduced into the laboratory for the identification and characterization of microbial pathogens. This unit contains examples of several basic approaches for microbial detection or characterization in the laboratory. Methods in this chapter include automated nucleic acid extraction, direct detection of microbial pathogens, and characterization of pathogens by DNA sequencing or typing. Any of these methods could be customized for the characterization of bacteria, fungi, parasites, or viruses.

Stochastic profiling of transcriptional regulatory heterogeneities in tissues, tumors and cultured cells

“…Genomic 23-26 and proteomic 27-30 methods are actively advancing, but the first to appear was single-cell transcriptomics 31-33 . The details of mRNA expression profiling in single cells can vary widely depending on the method 31-43 . Algorithmically, however, the different protocols all involve roughly the same steps: 1) extract cellular RNA by chemical, thermal, or enzymatic methods; 2) perform an oligo(dT)-based capture or an abbreviated oligo(dT)-primed reverse transcription (RT) to prepare a cDNA library of roughly uniform length; 3) tail the library with homopolymer; 4) exponentially pre-amplify the tailed cDNA with a universal homopolymer-containing primer; and 5) detect the amplification products by quantitative PCR (qPCR), oligonucleotide microarrays, or RNA-seq.…”

“…Interestingly, when similar approaches were applied to cells from a common lineage—where regulatory heterogeneities are possibly more quantitative rather than qualitative—the findings were much more generic 48-50 . This suggested that existing transcriptomic methods did not clearly separate biological variability from measurement variability when using one cell worth of starting material 42, 43 . Indeed, certain steps essential to the procedure, such as the RT step, are known to add substantial measurement variation when starting with minute amounts of input RNA 51, 52 .…”