Molecular Diagnostics for the Detection and Characterization of Microbial Pathogens

Abstract

New and advanced methods of molecular diagnostics are changing the way we practice clinical microbiology, which affects the practice of medicine. Signal amplification and real-time nucleic acid amplification technologies offer a sensitive and specific result with a more rapid turnaround time than has ever before been possible. Numerous methods of postamplification analysis afford the simultaneous detection and differentiation of numerous microbial pathogens, their mechanisms of resistance, and the construction of disease-specific assays. The technical feasibility of these assays has already been demonstrated. How these new, often more expensive tests will be incorporated into routine practice and the impact they will have on patient care remain to be determined. One of the most attractive uses for such techniques is to achieve a more rapid characterization of the infectious agent so that a narrower-spectrum antimicrobial agent may be used, which should have an impact on resistance patterns.

The Utility of Rapid Microbiological and Molecular Techniques in Optimizing Antimicrobial Therapy

“…This eliminates the time needed to obtain adequate bacterial growth that is required by current culture-based techniques. Polymerase chain reaction (PCR) based methods have been used in the research laboratory for more than two decades but have not been user friendly enough to be efficiently utilized in the clinical laboratory until 5-7 years ago [12]. PCR is considered to be a type of nucleic acid amplification technique that works by amplifying and measuring small specific sequences of DNA directly from a blood sample using fluorescence resonance emission transfer probes, molecular beacons, or Taqman probes [12].…”

“…Polymerase chain reaction (PCR) based methods have been used in the research laboratory for more than two decades but have not been user friendly enough to be efficiently utilized in the clinical laboratory until 5-7 years ago [12]. PCR is considered to be a type of nucleic acid amplification technique that works by amplifying and measuring small specific sequences of DNA directly from a blood sample using fluorescence resonance emission transfer probes, molecular beacons, or Taqman probes [12]. Each probe is designed to bind to a specific gene associated with the species of an organism, antimicrobial resistance (e.g., the mecA gene in methicillin-resistant S. aureus), or a subtype of these genes for epidemiological purposes (e.g., the mecA cassette IV associated with community-acquired S. aureus infection) [12].…”

“…PCR is considered to be a type of nucleic acid amplification technique that works by amplifying and measuring small specific sequences of DNA directly from a blood sample using fluorescence resonance emission transfer probes, molecular beacons, or Taqman probes [12]. Each probe is designed to bind to a specific gene associated with the species of an organism, antimicrobial resistance (e.g., the mecA gene in methicillin-resistant S. aureus), or a subtype of these genes for epidemiological purposes (e.g., the mecA cassette IV associated with community-acquired S. aureus infection) [12]. There are also probes available for detection of virulence factors (e.g., Panton-Valentine leukocidin cytotoxin commonly found in community-acquired methicillin-resistant S. aureus), providing more detailed information about the pathogen [12].…”

“…Each probe is designed to bind to a specific gene associated with the species of an organism, antimicrobial resistance (e.g., the mecA gene in methicillin-resistant S. aureus), or a subtype of these genes for epidemiological purposes (e.g., the mecA cassette IV associated with community-acquired S. aureus infection) [12]. There are also probes available for detection of virulence factors (e.g., Panton-Valentine leukocidin cytotoxin commonly found in community-acquired methicillin-resistant S. aureus), providing more detailed information about the pathogen [12]. When the probe binds to the amplified gene product, it creates a measurable amount of fluorescent signal that can be quantified exponentially by an RT-PCR machine [13].…”

“…It is similar to nucleic acid amplification techniques in that it uses fluorescein labeled probes, but it differs by binding to the 16 s rRNA of the live bacteria rather than binding to amplified gene products [12]. Once bound to the specific 16 s rRNA sequence, a fluorescent signal is created and the sample is read under a fluorescent microscope by a laboratory technician [15].…”