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Chapter 21 : Multiplex PCR Product Detection and Discrimination
Category: Clinical Microbiology; Bacterial Pathogenesis
This chapter examines the current state-of-the-art multiplex PCR and diagnostic platforms that are based on multiplex PCR but contain additional features to enhance sensitivity, multiplexing capability, or ease of use. Advances that aid in the development and optimization of multiplex rtPCR-based assays, such as primer design software and novel rtPCR reagents, are also reviewed. Multiplex PCR is a technique in which the amplification and detection of two or more target DNA or RNA sequences in a single reaction are accomplished through the use of specific primers or a combination of specific primers and probes. Microarrays have been utilized to measure the levels of expression of genes, to identify single-nucleotide polymorphisms, and to genotype organisms. Another technology that is being utilized is the reverse line-blot hybridization assay (mPCR/RLB). This method in combination with multiplex PCR has been reported to provide some benefits over microarray methods. Bead-based suspension array technology is being utilized currently for antigen-, protein-, and nucleotide-based detection assays. New technologies and new platforms for high-throughput DNA sequencing are reaching maturity and should soon be available for routine use in diagnostic laboratories. With concomitant advances in the fields of microfluidics, nucleotide and fluorescent dye chemistries, and information processing, highly multiplexed nucleic acid detection and identification technologies will gradually come to be applied in a vast range of situations in which sensitivity, specificity, and speed are indispensable.
FilmArray System from Idaho Technology. This system involves two stages of PCR utilizing a fixed array and can analyze 120 different targets per sample. This figure was compiled from information obtained at the Idaho Technology website (www.idahotech.com). Reagents for this system can be stored at room temperature, and controls for extraction and PCR are performed with each assay; the run takes approximately 1 h from sample injection into the test pouch.
DHPLC. The technology identifies mutations and polymorphisms based on detection of heteroduplex formation between mismatched nucleotides in PCR-amplified DNA. Sequence variation creates a mixed population of heteroduplexes and homoduplexes during reannealing of wild-type and mutant DNA. These complexes are detected by the alteration in the banding patterns seen upon analysis by the DHPLC instrument.
Schematic representation of MassTag PCR.
rtPCR platforms available for multiplexing
Software packages available for multiplex PCR assay development a
Multiplex PCR master mix kits a
Commercially available multiplex kits or assays a