Molecular assays hold huge potential for future diagnostics

The discovery of polymerase chain reaction (PCR) in 1985 was an important milestone. The objective was to improve the synthesis of oligonucleotides. But over the last three-and-a-half decades, PCR has not only proved its worth in research and development (R&D) but has also become an indispensable tool in the field of medical diagnostics. This can be appreciated by the prolific technological advancements of PCR applications in both the domains. In parallel, our comprehension of the PCR technology has also expanded and that has allowed us to rationalize its employment. We now see PCR being used as a fast alternative to gene cloning; for manipulations of DNA fragments; for detection of pathogenic microorganisms with higher sensitivity and specificity and, if desired, followed by an accurate genotyping; archaeological specimens DNA analysis; identification of mutations relevant for inherited disorders, disease predisposition and malignant transformation; perform HLA typing; for forensic applications like paternity testing; understanding evolution; and getting insights into differential gene expressions.

Advancements in molecular diagnostics have played an important role in the improvement of patient outcomes by refining the disease diagnosis, indicating prognosis, supporting theranostics and allowing us to track treatment effectiveness. The emergence of Covid-19 and successful use of real-time PCR in Covid diagnostics has realigned the focus of medical diagnostic industry on molecular assays. The world is observing a dramatic upsurge in the R&D efforts to make best use of the intrinsic capabilities of PCR technology by combining it with innovative solutions to feed the rising demand for sensitive, specific, rapid diagnostic solutions for early-stage detection of acute as well as chronic illness in both infectious as well as non-infectious domains.

The use of real-time PCR for qualitative and quantitative analysis of common pathogens has become unexceptional across conventional labs. It allows high throughput and rapid turnaround time. These capabilities of RT-PCR along with other factors have ushered in a demand and adoption of culture independent diagnostic testing assays such as syndromic multiplex testing. Multiplex testing allows simultaneous detection of most common pathogens capable of causing a specific syndrome in one panel. This can significantly reduce the time needed to arrive at an accurate and effective diagnosis. Upcoming technologies like ddPCR share unprecedented potential in the field of absolute quantification of the specified targets in low-volume samples. The technology allows the user to analyze gene mutations in low-tumor-load samples, rare allele targets, and low-pathogen-load specimens. Similarly, technologies like Immuno-PCR (iPCR), high-resolution melting analysis (HRM), immuno-RCA (immunoassay with rolling circle DNA amplification), proximity ligation assay (PLA), transcription-mediated amplification (TMA), reverse line blotting assays (RLBA), single-cell PCR, clustered regularly interspaced short palindromic repeats (CRISPR), gene chip technology, electrochemical proximity assay (ECPA), loop-mediated isothermal amplification (LAMP), single-tube multiplex amplification in real-time (SMART), Sanger sequencing, and high-throughput sequencing technology like next-generation sequencing (NGS) and micro-array technologies are making significant inroads into medical diagnostics.

Even though emerging nucleic acid amplification technologies (NAAT) have the potential to improve sample to answer response time and provide personalized responses, still further improvements are needed. The need of the hour is to integrate the whole sample processing flow (sample extraction to PCR amplification) using the innovative microfluidic designs into a compact and more flexible package in terms of ease of use to accelerate treatment choice as well as expedite operational decision making and explain resource utilization. With the discovery of newer biomarkers and upgradation of NAAT devices, molecular diagnostics has the capability to provide novel tools to further facilitate precision diagnostics and help in successful individualized therapeutic interventions.