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Chapter 16 : Microwell Plate Detection Systems for Amplicon Detection and Characterization
Category: Clinical Microbiology; Bacterial Pathogenesis
Sequence-specific methods for the detection of the products of nucleic acid amplification procedures (amplicons) have been developed for a variety of solid phases, including nylon membranes, microwell plates, microparticles, and, most recently, microchips (oligonucleotide probe microarrays). This chapter deals with procedures in which the molecules that capture amplicons are immobilized onto the surface of wells of microwell plates. Microwell plate detection systems can be divided into two formats based on the molecule used to capture amplicons: an oligonucleotide probe (sequence-specific capture) and avidin (nonspecific capture). Microwell plate detection systems have been developed in-house and are available commercially. Microwell plate detection procedures are frequently based on capture and detection of biotinylated amplicons. This requires that the primer used in the PCR amplification to generate the amplicon strand complementary to the probe be tagged with biotin. The biotin substituent will allow the amplicon to be either captured or detected by avidin (or streptavidin), depending on the detection format. Polystyrene microwell plates are typically used for amplicon detection because of their high DNA binding capabilities. There is substantial literature demonstrating the excellent analytical performance and clinical utility of microwell plate detection systems for PCR amplicon detection. Microwell plate detection of amplicons is flexible, is compatible with virtually any target, and can easily detect multiple pathogens under common hybridization and wash conditions.
Schematic diagrams of microwell plate detection using sandwich (A) and direct (B and C) hybridization formats.
Conditions for immobilization of DNA probes in microwell plates a
Microwell plate hybridization conditions a
Advantages and disadvantages of microwell plate detection systems versus alternatives