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Chapter 15 : Detection of Pathogenic Organisms with Multicolor Molecular Beacons
Category: Clinical Microbiology; Bacterial Pathogenesis
The chapter describes a detection method that is based on the sequence-dependent hybridization of fluorogenic reporter molecules called “molecular beacons’’ and its applications. Molecular beacon probes represent a new class of oligonucleotides that can hybridize and report the presence of specific nucleic acids in homogeneous solutions. In addition, improvisations on the basic theme of molecular beacons have also appeared in the literature. Molecular beacons are primarily employed as highly specific amplicon detection probes in homogeneous, real-time, multiplex gene amplification assays. On the other hand, the generation of all five fluorescent colors during PCR amplification indicates that the mycobacteria in the sample are rifampin susceptible. Infection by certain types of human papillomavirus (HPV) can lead to cervical cancer; therefore, accurate identification of these oncogenic HPV genotypes is critical. One assay detects HPV-DNA by SYBR Green® and then distinguishes the seven most prevalent high-risk HPV genotypes by using real-time molecular beacon PCR. They can be used as hybridization detection probes, not only for real-time monitoring of DNA amplification in vitro but also for real-time monitoring of the distribution and transport of mRNAs in living cells. The current available applications of molecular beacons for detection of pathogenic microorganisms are listed in the chapter. Efforts are being made to explore their applications in many areas including genotyping of infectious agents and mutation analysis for the identification of drug-resistant pathogens.
Principle of operation of molecular beacons. The hairpin stem formed by the complementary arm sequences cannot coexist with the rigid double helix that is formed when the probe hybridizes to its target. Consequently, the molecular beacon undergoes a conformational change that forces the arm sequences apart and causes the fluorophore to move away from the quencher.
Comparison of hybridization detection assays
Comparison of differently structured probes in real-time PCR assay
Applications of molecular beacons for pathogenic organisms a