PCR Methods for Microbial Diagnostics

In vitro amplification of a pathogen-specific nucleic acid sequence by polymerase chain reaction (PCR) allows rapid diagnosis with a high degree of sensitivity and specificity. Over the past 10-15 years, there has been increasing use of nucleic acid amplification tests in the routine diagnosis of infectious disease [31]. PCR technology is applicable to virtually any bacterial pathogen and is commonly used along with pulsed-field gel electrophoresis (PFGE) in molecular epidemiology of bacterial pathogens [32]. The ability to include virtually all known sequence targets, the enormous sensitivity, and the application of the technique in various diagnostic settings has contributed to the widespread acceptance of PCR as the standard method for detecting nucleic acids from a number of sample and microbial types [33]. Conventional PCR is an important tool for pathogen detection, but it has not been possible to accurately identify PCR products without sequencing, digestion by restriction enzymes, or Southern blotting. The more recent development of real-time PCR applications has gained wider acceptance because it is more rapid, sensitive, and reproducible, while the risk of carryover contamination is minimized [34, 35]. An increasing number of chemistries are used to detect PCR products as they accumulate within a closed reaction vessel during real-time PCR, including the nonspecific DNA-binding fluorophores and the specific fluor-ophore-labeled oligonucleotide probes [36].

In addition to molecular typing methods, growing knowledge of different pathogenic traits and genetically distinct pathotypes offers the opportunity to unravel the pathogenic fine structure of bacterial isolates obtained from diagnostic samples. Virulence genes or gene clusters have been characterized in numerous pathogenic bacteria, leading to a complex picture of virulence traits in several bacterial species [37].

Besides the identification and classification of bacterial pathogens, the detection of specific antibiotic resistance in clinical isolates has always been the second major task of microbiological diagnostics. In recent years the molecular mechanisms of antibiotic resistance have been thoroughly characterized, providing the basis for molecular tool to detect a multitude of antibiotic resistance mechanisms on the level of chromosomal or episomal DNA [34, 38, 39]. The introduction of real-time PCR methods offers a cost-effective, user-friendly format for genetic methods that fuels their use for the detection and characterization of antimicrobial resistance determinants in routine diagnostic microbiology. The implementation of these assays to detect resistance in clinically important slow-growing organisms (e.g., Mycobacterium tuberculosis), to rapidly identify clinically important resistance mechanisms, and to overcome laborious and time-consuming culture techniques in the control and surveillance of methicillin-resistant Staphy-lococcus aureus (MRSA) and glycopeptide-resistant enterococci (GRE) carriage is of particular interest. For reference laboratories it is important to have a broad repertoire of genetic assays to confirm defined resistance determinants, to sort out ambiguous phenotypic results, and to provide a reliable scientific basis for molecular surveillance of antimicrobial-resistant bacteria and resistance determinants in a global network.

Thus, molecular testing methods based on DNA/RNA amplification have the potential to replace many conventional microbiology laboratory assays. Although the molecular techniques in microbial diagnostics may be unlikely to replace culture for normally sterile specimens, e.g., blood or cerebrospinal fluid, they are particularly useful for identifying specific pathogens against the background of a complex mixed microflora. Quality criteria include appropriate specimens for analysis, performance characteristics of different analytical methods, optimal specimen processing, the effects of PCR inhibitors, and false-positive results caused by contaminating nucleic acids. Recent refinements in PCR technology have resulted in more user-friendly testing platforms. These platforms are automated and have lowered risks for contamination, decreased costs, and are faster than former platforms. Additionally, the extreme sensitivity of these techniques coupled with the potential presence of small numbers of pathogenic organisms in asymptomatic individuals should be considered carefully. However, as DNA-based molecular detection techniques do not generally have the ability to determine whether an organism is dead or alive, these techniques have limited benefit in monitoring therapy, e.g., in infections due to M. tuberculosis. Quality control guidelines for molecular microbiological diagnostic assays are in their infancy and require further development. Finally, a major drawback of today's PCR-based methods in microbiological diagnostics is that parallel testing in a single step is usually restricted to a limited number of genes, making these methods inappropriate for simultaneous scanning of large quantity of diagnostic items.

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