Molecular Diagnostics (MDx) is a field of laboratory medicine which applies molecular biology techniques to study human disease. MDx applications target sequences of human and microbial genomes, as well as the proteins they encode, to assess a patient’s health at the molecular level. Such methods can be used to identify genetic biomarkers, germline and somatic variations, infectious pathogens, and monitor progression of disease. Since the dawn of recombinant DNA technology, monumental accomplishments in life science technologies such the invention of polymerase chain reaction (PCR), breakthroughs in next-generation sequencing, and the completion of the Human Genome Project, have accelerated this field of study. As innovation marches on, innovative technologies continue to push towards more accurate, rapid, cost-effective, and high-throughput methods for molecular analysis and diagnosis.
The COVID-19 outbreak accelerated MDx innovation by bridging basic research and clinical reality with new devices and technologies to answer urgent needs in clinical management. The pandemic created a nearly universal acceptance of MDx at various levels, from global health organizations, public and private companies, schools, hospitals, physicians, and patients to anyone wanting a fast and reliable COVID-19 test result. Consumers are now more educated; terms like PCR, antigen tests, and antibodies are widely understood. A “diagnostic consumerism” has begun as patients not only expect easy access to testing but also transparency in how the test works and what the results mean.
With so many new developments, understanding the differences and nuances between MDx technologies and platforms can be daunting – even for healthcare professionals. This article takes a closer look at some of the most widely utilized molecular genetic testing technologies, their workflows (Figure 1) and applications.
Figure 1 – Schematic workflow of qPCR and NGS testing
Nucleic acid isolation
Nucleic acid extraction is generally required for both sequencing as well as NAAT (Nucleic Acid Amplification Tests) testing, as the targeted genetic material is often embedded in cells, viral particles, or otherwise shielded in a way that interferes with the test. Cells, proteins and other molecules in the collected specimen milieu or the collection/transport medium can also contain inhibitors. Sample preparation is therefore a critical step for most MDx techniques, and the extraction technology must be suitable for the given sample type (blood, urine, feces, saliva, etc.) as well as the designated nucleic acid (DNA, RNA, or both).
Sequencing
Sequencing is a technology that deciphers genetic information and determines the identity and order of nucleotides in DNA. The first milestone was the development of “Sanger sequencing”, a targeted approach with one sequence per reaction. The next breakthrough came with the development of next generation sequencing (NGS) technologies. The term “NGS” covers a variety of different detection chemistries and workflows, each with its advantages and trade-offs. The second generation of sequencing undertakes a massively parallel sequencing approach which involves library preparation and target enrichment prior to sequencing. Some popular NGS chemistries are sequencing-by-synthesis, semi-conductor sequencing or measurement of electrical current as nucleic acids pass through a nanopore. The turnaround time from sample to result can vary depending on the platform and application and is often between one to several days but can also take more than a week.
NGS offers comprehensive genomic profiling, pathogen identification, and antibiotic resistance gene detection. NGS can be targeted, covering selected fragments of hundreds of pathogens and/or genetic biomarkers simultaneously. Alternatively, NGS can be untargeted – using whole exome or whole genome sequencing. Metagenomic NGS (mNGS) is an untargeted approach in which all the nucleic acid in a sample is sequenced. This provides the most comprehensive genetic information of a patient sample and allows the identification and characterization of previously unknown emerging pathogens.
NGS is recognized as an important in vitro diagnostic technology. In 2018, the Food and Drug Administration (FDA) issued guidance on the development and validation of NGS-based IVDs intended to aid in the diagnosis of suspected germline diseases. The Centers for Medicare & Medicaid Services (CMS) determined coverage for patients with somatic cancer in 2018 and for patients with germline cancer in 2020. There are currently a number of FDA approved IVDs on the market.
Besides cancer, there are many other disease areas that benefit from comprehensive genetic profiles. For example, the World Health Organization has called out the potential of NGS in diagnosing drug-resistant tuberculosis, but also highlighted challenges such as workflow integration and training, cost and reimbursement, and interpretation of sequencing data. Other areas that benefit from NGS include non-invasive prenatal testing and antibiotic resistance testing in infectious diseases. Sequencing played a key role in the early identification and characterization of SARS-CoV-2 as pathogen causing COVID-19 and in the continuous genomic surveillance of the virus. However, for most routine diagnostics, the cost and turnaround time of sequencing can be prohibitive.
Nucleic Acid Amplification Tests
PCR uses sequence-specific primers to amplify a few copies of DNA to millions, or even billions of copies. When targeting RNA, the RNA is first reverse-transcribed into a complementary DNA (cDNA) strand prior to PCR amplification. In real-time PCR, also known as quantitative PCR (qPCR), an additional sequence-specific probe leads to the emission of a fluorescence signal when the target is present and amplified. The fluorescent signal intensity enables a highly accurate quantification of the amount of target in a sample relative to a standard with defined target amounts. One hallmark of PCR is that the reaction cycles between different temperatures. PCR run times vary from 90-120 minutes on average. By utilizing different fluorochromes, qPCR supports multiplex testing that enables the detection of multiple targets in a single reaction. Using the same sample in different qPCR reactions allows for syndromic panel testing.
The FDA has approved PCR-based IVDs for a variety of applications, including human genetic, microbial and companion diagnostic tests. PCR is widely used and accepted for infectious pathogen detection and utilized in response to COVID-19, including Emergency Use Authorization (EUA) for at the point of care testing. Taking advantage of its multiplex capability, multiple tests target SARS-CoV-2 and influenza A and B in parallel.
Digital PCR (dPCR) allows for the absolute quantification of a target without signal-comparison to a standard. Besides absolute quantification, dPCR enables the detection of mutations with very low frequencies or copy number variations (CNVs). Isothermal amplification methods are designed to amplify the targeted sequence at one constant temperature, which allows for extremely fast continuous amplification. Isothermal amplification assays can be designed with a visual read-out for easy employment at low-resource settings. In general, isothermal amplification is a promising technology for diagnostic devices at the point of care (POC) and over the counter (OTC) at-home settings. In response to COVID-19, several tests based on isothermal-amplification received EUA, including OTC at-home tests.
Conclusion
MDx has become indispensable in the clinical environment. Physicians can now choose from an ever-growing test menu, with testing performed at clinical laboratories using a wide variety of platforms. Realizing that patients are molecularly unique, MDx is critical for personalized, tailored treatments. While NGS is widely used to detect and characterize new pathogens and emerging mutations in the field of clinical microbiology, oncology-focused NGS solutions are crucial in the diagnostic world and offered by many CLIA-certified laboratories. Meanwhile, NAAT tests such as PCR or isothermal amplification offer reliable and sensitive detection of known pathogens or disease biomarkers. They have faster turnaround times and lower price-points than sequencing. Many qPCR-based assays are scalable to support higher throughput testing during peak times, while multiplexing options support syndromic testing.
MDx has transformed medicine since its development a few decades ago (Figure 2). These days, physicians can use MDx to quickly identify disease-causing organisms and molecular variations in cancer genomes to make more effective treatment decisions. Compared to more traditional clinical methodologies, molecular diagnostic testing results in faster turnaround times, higher sensitivity, and higher specificity, which can ultimately reduce costs and save lives.
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