In vitro diagnostics (IVD) is an umbrella term for tests conducted on blood or tissue samples to detect diseases, determine the efficacy of novel or established treatments, and monitor health. Emerging precision medicine approaches use IVDs to identify patients who are likely to benefit from a specific treatment.
Overall, IVDs can be classified into immunological, chemical, microbiological, molecular, and organ-on-chip assays. Whereas immunological IVDs, such as ELISAs (enzyme-linked immunosorbent assay), RIAs (radioimmunoassay), and lateral flow assays, take advantage of highly specific antibody-antigen interactions, chemical IVDs are essentially medical devices that employ specific biochemical reactions to detect proteins or metabolites, such as at-home glucose, hemoglobin, or cholesterol testing kits.
Microbiological IVDs culture specific bacteria or fungi to detect infections from blood, stool, or throat swabs samples, while molecular IVDs detect DNA or RNA sequence signatures of diseases or pathogens in patient samples. Recent innovations in microfluidic organ-on-chip technology are adding yet another new dimension to IVDs where entire organs are modeled on microfluidic chips to test drug toxicity, efficacy, and permeability before animal model and human studies.1
In addition to the spurt of molecular diagnostics in point-of-care (POC) tests triggered by the COVID-19 pandemic, in recent years molecular IVDs have expanded beyond the detection of infectious agents to diverse biomedical fields including oncology, hematology, genetics, clinical chemistry, and patient-tailored precision medicine interventions. Antibody-based ELISA tests remain the gold standard for confirming the presence of pathogens, but nucleic-acid-based tests can detect infectious agents earlier and with high sensitivity. Multiplex quantitative PCR, reverse transcription PCR, and reverse transcription isothermal amplification are widely regarded as optimal confirmatory tests to detect viral RNA. With a market currently estimated at $89 billion and expected to reach $118 billion by 2027, molecular diagnostics is the most rapidly growing sector in IVDs.
Gerald Hunter, PhD, field application scientist at Fortis Life Sciences, has extensive experience in developing nucleic acid-based assays. “PCR-based diagnostics offer several advantages over conventional diagnostics such as speed, sensitivity, specificity, multiplexing capabilities, and the capacity to be automated within a diagnostic workflow,” said Hunter.
Constellation of PCR-based techniques
Although there are a wide variety of nuanced methods that rely on PCR, such as digital-, inverse-, amplified fragment length polymorphism (AFLP)-, end-point-, hot-start-, in situ-, real-time-, solid-phase-, touch down-PCR, all PCR techniques use primers or short synthetic single-stranded oligonucleotide fragments of complementary DNA that bind to templates to amplify defined sequences million or billion folds during successive rounds of denaturation, annealing, and polymerization. Each step is initiated by the cyclical changes of temperature in an instrument called the thermal cycler which is a staple in almost every laboratory.
While traditional end-point PCR detects the presence or absence of a specific DNA sequence, its readout is purely qualitative. When the amount of end-point product is compared to that of a housekeeping or control gene, the method yields semi-quantitative results.
Hunter said, “Semi-quantitative PCR is used mainly to determine if a gene is upregulated or downregulated in the presence of a stimulus.”
Quantitative PCR shows the increase in nucleic acid amplification over time, and is used in gene expression analysis, genotyping single-nucleotide polymorphisms (SNPs) or copy number variations (CNVs), detecting pathogens, and monitoring therapeutic efficacy. Quantitative reverse transcription PCR (RT-qPCR) uses RNA as the starting material, which is first transcribed into complementary DNA (cDNA) by a reverse transcriptase enzyme. The cDNA template is then amplified using qPCR. Real-time quantitative PCR uses fluorescent reporter molecules that yield an increasing amount of fluorescence with an increase in the amount of amplified DNA product and requires a specialized thermocycler.
“Quantitative PCR (qPCR or RT-PCR) is widely used to determine the quantity of target DNA in a sample. However, a standard curve of serially diluted standard samples must be used to make this determination,” said Hunter.
Other common upgrades to conventional PCR include hot-start and multiplex PCR. Hot-start PCR uses a modified polymerase that reduces the nonspecific amplification of unintentional products and the generation of primer dimers at low temperatures by maintaining the polymerase in a nonfunctional state until heat activation. Multiplex PCR amplifies multiple targets in the same reaction tube, with a different pair of primers for each non-overlapping target. This requires designing distinct primers that amplify products of different sizes that can be detected and distinguished simultaneously, using agarose gel electrophoresis. Multiplexing saves resources from sampling to diagnosis.
Multiplexing advantage in PCRs
Molecular diagnostics span the spectrum of workflow times and multiplexing capacity.2 Unlike PCR, isothermal amplification (IA) involves continuous exponential amplification of nucleic acids at a constant temperature using enzymes such as strand-displacement polymerases rather than cyclical changes in temperature. Both qPCR and IA have limited multiplexing capabilities, but they also have shorter workflows compared to other molecular diagnostic techniques such as in situ hybridization (ISH), with the added potential for automation to decrease turnaround times.
ISH involves the binding of labeled probe sequences to DNA or RNA in tissue samples. The technique requires considerable hands-on time and extensive optimization that leads to low multiplexing capabilities, procedural errors, and long turnaround times.
The other end of the molecular diagnostics spectrum is dominated by microarrays and Sanger and next-generation sequencing techniques with high multiplexing capabilities but the inherent drawback of higher procedure times. While microarrays can detect thousands of genes simultaneously (which is not possible through PCR), Sanger sequencing is a targeted sequencing technique that uses oligonucleotide primers to sequence specific regions of DNA and massively parallel deep sequencing, or next generation sequencing (NGS) can sequence the entire human genome in a single day or less.
There are no one-size-fits-all solutions when it comes to molecular diagnostics. Therefore, it is important to consider the availability of time versus multiplexing capabilities to select an optimal technique.
PCR versus isothermal amplification
Isothermal amplification methods include strand displacement amplification, rolling cycle amplification, whole genome amplification, loop-mediated isothermal amplification (LAMP), helicase-dependent amplification, and multiple displacement amplification among others. The reaction products can be used in subsequent applications such as lateral flow, for the detection of the product.
“Isothermal amplification is ideal for situations in which there is no access to a thermal cycler (e.g., clinics, field work, home testing kits), and can sometimes be faster, simpler, and more cost-effective if the diagnosis only requires a quick qualitative answer.”
Essentially, when a quick qualitative answer is the desired endpoint, IA can be the more judicious and cost-effective option, despite lower sensitivity or specificity, compared to PCR. COVID-19 testing has employed IA. For example, a 30-minute, single-tube, colorimetric RT-LAMP test for COVID-19 that uses nasopharyngeal swabs or saliva samples is based on IA. It is suitable for POC or low-resource settings as it uses resources unlikely to encounter bottlenecks. While not as sensitive as RT-PCR tests, RT-LAMP tests are suitable for the surveillance and screening of infectious individuals who are asymptomatic or pre-symptomatic.
Temperature changes are the main difference between PCR and isothermal amplification methods. In PCR, a thermocycler changes the reaction temperatures cyclically to influence the actions of the temperature dependent reagents. In contrast, isothermal amplification occurs at a single temperature. Instead of using heat to unzip or denature DNA as in PCR, isothermal amplification uses strand displacement polymerases to unzip the DNA double helix as the enzyme moves forward at a constant temperature.
The choice between PCR and IA depends on the application and the available instruments. Whereas the readout in qPCR is discrete Ct values, IA provides fluorescence values at certain time intervals. While PCR is common, it requires access to a reliable thermal cycler, which is not possible for POC diagnostics.
“This can be a disadvantage or even a deal-breaker for point-of-care and diagnostic applications,” said Hunter. “If a thermal cycler is available, PCR amplification is a relatively straightforward process, easier to design and optimize, and can be a better choice for quantifying the detection of rare transcripts.”
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- Khodakov D, Li J, Zhang JX, Zhang DY. Highly multiplexed rapid DNA detection with single-nucleotide specificity via convective PCR in a portable device. Nat Biomed Eng. 2021 Jul;5(7):702-712. PMID: 34211146.
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