PCR-Based Assays for Antibiotic Susceptibility Testing
Knowledge of the molecular determinants of antibiotic resistance has led to the development of new methods for the rapid detection of resistance in bacterial pathogens. PCR-based techniques, microfluidics, microarrays, mass spectrometry, cell lysis based methods, and whole-genome sequencing have all demonstrated the ability to detect resistance in various bacterial species. Creative Biolabs is a premier provider of customized solutions to live biotherapeutic product (LBP) development. Our deep industry expertise coupled with a multi-platform approach delivers tailored antibiotic susceptibility testing (AST) for LBP.
PCR-Based Techniques
PCR-based techniques (both conventional and real-time) rely on the sequence-specific amplification of nucleic acids. PCR is one of the most efficient and rapid molecular tools for quantifying and analyzing bacterial infection genes. The general approach to PCR involves denaturation of the primers, annealing, and extended circulation of the primers by heat-resistant DNA polymerase in a compatible buffer containing nucleotides, ions, etc. DNA analysis methods such as electrophoresis, southern blotting, restriction fragment length polymorphism, single-stranded conformation polymorphism, DNA fingerprinting and molecular beacons can be used to determine whether the amplified target has resistance genes. Another tool developed based on PCR is LAMP, which is also used for AST evaluation.
PCR-Based Assays for AST
With increased knowledge of the genetic basis of antibiotic resistance, PCR-based approaches have been developed for detecting the presence of genetic determinants of resistance to a variety of antibiotics for many different bacterial species. A notable example is the use of PCR to identify methicillin-resistant Staphylococcus aureus (MRSA) by detecting the mecA gene. PCR-based methods have also been developed to detect vancomycin resistance associated with the vanA and vanB genes and have been used primarily to detect resistance in enterococcus. Some earlier DNA probe tests for resistance genes have been replaced by novel PCR assays. Because real-time fluorescence quantitative PCR can provide accurate information on the copy number of the genome, a very short incubation time can be used to distinguish susceptible and resistant strains. One advantage of this approach over the PCR-based approach described above is that it does not rely on the mechanism of resistance and indirectly measures phenotypic resistance by detecting growth in the presence of antibiotics.
Fig.1 Digital PCR-High Resolution Melt analysis (HRM)-based bacterial identification from mixed bacterial samples. (Khan, 2019)
Advantages and Disadvantages
The main advantage of these PCR-based methods is that they can be performed in a relatively short time and, in some cases, pure cultures are not required to use clinical samples. Thus, PCR has the potential to significantly reduce turnaround time and rapidly provide information on antibiotic resistance. However, the main limitation of this method is that the presence of resistance genes may not always be associated with phenotypic resistance.
AST continues to be the most functional and globally accepted reference method in guiding antimicrobial therapy. Genotypic PCR-based assays are revolutionary to clinical laboratory testing and epidemiologic monitoring because of the rapid detection of resistant organisms with greater accuracy than phenotypic testing. At Creative Biolabs, delivering the best products and services is just the start; we make sure each client gets our utmost attention and each project benefits from a customized service plan. If you are interested in our PCR-based assays for antibiotic susceptibility testing services, please contact us to get started.
Reference
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Khan, Z.A.; et al. Current and emerging methods of antibiotic susceptibility testing. Diagnostics. 2019, 9(2): 49.
For Research Use Only. Not intended for use in food manufacturing or medical procedures (diagnostics or therapeutics). Do Not Use in Humans.
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