PCR and molecular diagnostics: Biotechnologic integration in clinical laboratories

A new generation of molecular tests has revolutionized laboratory medicine through allowing for quick, sensitive, and precise identification of the pathogen; genetic mutation or alteration; and disease-related bio-markers. Of all the molecular techniques that exist today, PCR is still the most versatile, and clinically applicable technique available. This paper reviews the development of PCR technology from traditional amplification to advanced platforms including qPCR, dPCR, RT-PCR, and multiplex PCR. Each of the advanced platforms reviewed will be examined for their contributions to both the accurate detection, and quantification of nucleic acids along with the ability to perform high throughput analyses. Additionally, this paper also discusses how PCR technology can be integrated with additional technologies such as NGS, CRISPR-based diagnostic systems, microarrays, and epigenetic profiling to provide a larger view of disease states than was previously possible. Specifically, the focus of this paper will include many clinically relevant biomarkers and molecular targets used in cancer research including EGFR, KRAS, BRAF, BCR-ABL1, circulating tumor DNA (ctDNA), and methylated SEPT9. These biomarkers and molecular targets have greatly enhanced early diagnosis, prognostication, treatment monitoring and precision medicine in cancer research and other areas. The use of PCR-based technologies in diagnosing infectious diseases, tracking AMR, detecting heritable disorders, studying microbial communities, and MRD monitoring were also discussed. Further discussion included current trends in integrating multiple omics data types into a single platform using AI assisted data analysis tools. Liquid biopsy technologies and point-of-care molecular test platforms were also discussed. Although there are continuing challenges related to costs associated with developing these technologies, establishing standardized protocols for each technology type developed, having skilled technicians develop and validate these technologies, and managing large amounts of data generated when using these technologies; continued advances are providing greater access to these technologies to clinicians and ultimately patients.