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Chapter 2 : DNA Probes for Culture Confirmation and Direct Detection of Bacterial and Fungal Infections: a Review of Current Technologies and Assays
DNA Probes for Culture Confirmation and Direct Detection of Bacterial and Fungal Infections: a Review of Current Technologies and Assays, Page 1 of 2< Previous page Next page > /docserver/preview/fulltext/10.1128/9781555816834/9781555814977_Chap02-1.gif /docserver/preview/fulltext/10.1128/9781555816834/9781555814977_Chap02-2.gif
This chapter focuses on nonamplified nucleic acid probes and their current uses in the clinical laboratory. DNA probes are pieces of nucleic acid that are labeled in some way and are designed to seek out and bind to stretches of DNA or RNA that have sequences that are complementary to the probe. In hybridization reactions, a double-stranded DNA molecule is denatured to single strands. Several formats for the hybridization reactions exist: solid phase; in solution (liquid phase); in situ; or by use of a Southern hybridization procedure after gel electrophoresis. In sandwich hybridization assays, one probe is attached to a solid support such as a nitrocellulose filter in single-stranded form and ‘‘captures’’ homologous nucleic acids in liquid samples; a second probe, which recognizes a contiguous area of the nucleic acid, carries the reporter molecule such as a radioisotope or biotin. The target and probe nucleic acids are free to move in solution, maximizing chances that complementary sequences will bind. Southern hybridization involves using purified DNA that is cleaved with restriction endonucleases. There are numerous methods for detecting the binding of probe to target nucleic acid. Commercially available DNA probes used for culture confirmation of bacteria, mycobacteria, and fungi are discussed in the chapter. Of the assays discussed in the chapter, probes for the detection of mycobacteria have had the greatest clinical impact. The chapter describes the utility of nonamplified probes for the diagnosis of sexually transmitted diseases, vaginal infections, and streptococcal pharyngitis.