Real-Time Polymerase Chain Reaction (real-time PCR) has revolutionized the diagnosis and management of infectious diseases in acute care settings. Through the coupling of the amplification and direct detection of target microbial DNA in a single reaction vessel, real-time PCR drastically reduces the need for microbial culture methods, decreases wait times required for pathogen detection, and allows clinicians to perform early targeted therapeutic interventions.
How Real-Time PCR Works
PCR amplifies short regions of DNA in vitro using an enzymatically-driven process. It relies on knowing partial sequences of DNA for microbes and developing oligonucleotide primers that hybridize specifically to these target sequences. The PCR method uses multiple cycles of heating and cooling to perform rounds of target DNA denaturation, primer hybridization, and primer extension using a thermostable DNA polymerase enzyme. As a result, the target DNA is amplified exponentially, potentially generating billions of copies of target DNA from a single copy in less than an hour. Technological advances to this breakthrough process have allowed increased sensitivity and specificity of detection through double amplification, simultaneous detection of target sequences through multiplex PCR, and evaluation of RNA viruses through reverse transcriptase PCR (RT-PCR) which involves the conversion of RNA into complimentary copies of DNA before amplification. The most significant of these improvements is real-time PCR, in which amplification and detection of target microbial DNA are carried out concurrently, providing speed, simplicity, and reproducibility to the entire process.
Real-Time PCR Use in Infectious Diseases
Real-time PCR is an indispensable tool used by infectious disease specialists due to its sensitivity, specificity, and fast turnaround times for pathogen detection, especially for organisms that cannot be grown in vitro or those with prolonged culture procedures that delay timely diagnoses. For example, detection through real-time PCR of Mycobacterium tuberculosis, a fastidious organism notorious for being slow-growing and difficult to culture, has revolutionized tuberculosis diagnosis and treatment programs worldwide with results released within two hours as compared to culture-based methods which takes weeks before results are released. Moreover, resistance to antimicrobial agents can be detected through real-time PCR, as in the case of the detection of mutations in the rpoB gene heralding resistance of tuberculosis strains to rifampicin, an extremely effective first-line anti-TB drug. The use of real-time PCR to rapidly detect antimicrobial-resistant strains of pathogens such as Pseudomonas aeruginosa, Helicobacter pylori, and Mycoplasma pneumoniae is increasingly becoming crucial for infectious disease specialists managing high-acuity patients in emergency departments and intensive care units and aiding in the delivery of targeted pharmaceutical interventions.
Finally, real-time PCR has also been used to accurately identify previously-unknown virulent pathogens. A real-time reverse transcription PCR (RT-PCR) assay of samples from severe pneumonia patients were instrumental in the detection and isolation of a novel coronavirus in 2019, which we now know as SARS-CoV-2, the etiologic agent of the COVID-19 pandemic. Real-time PCR assays have also been successfully used to identify SARS-CoV-2 variants, targeting the specific spike gene mutations of the Alpha, Beta, Delta, Gamma, and Omicron variants with near-identical results to the more costly and technologically-complex method of whole genome sequencing. Indeed, even with the wealth of diagnostic tools that have been developed to rapidly identify and detect the SARS-CoV-2, real-time PCR testing remains to be the preferred initial diagnostic test for patients suspected of having COVID-19 due to its high accuracy and quick time-to-diagnosis.
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Real-time PCR testing has proven time and again to be a powerful tool to have among the arsenal of infectious disease specialists and front-line physicians. It provides quick-but-accurate diagnoses that allows rapid triaging of patients; provides information on antimicrobial resistance and ensures early targeted antimicrobial therapy; and reduces repetitive testing and waiting times which provides substantial benefits to patients and healthcare providers alike.
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