Across all areas of life sciences research, scientists are increasingly aware of the need for absolute quantification of nucleic acids. To make their analyses actionable, scientists want to directly measure copies of a molecule, rather than relying on a simple positive or negative result. The challenge is that any tool for quantifying nucleic acids needs to be standardized against a universal reference material produced by an authoritative body such as the World Health Organization. If this practice is not in place, the results obtained using this tool will not be reproducible across different tests, laboratories, or locations.
Currently, there are no convenient primary reference measurement procedures for quantifying these standards. As a result, it is difficult for research laboratories and diagnostics manufacturers to harmonize their nucleic acid quantification methods and produce reliable assays. Fortunately, several metrology laboratories—laboratories that study the science of measurement—are seeking to develop a primary reference procedure using digital PCR technology. This technique provides absolute quantification of nucleic acid samples without the need for calibration.
Reliable RNA and DNA quantification
One area where absolute RNA and DNA quantification would be especially useful is in clinical laboratories. To create a reliable in vitro diagnostic, for instance, it needs to be calibrated against a standard that can be traced back to a Système International (SI) unit. This allows researchers to compare the resulting data directly against different laboratories to ensure accurate interpretation.
Creating a traceable diagnostic starts with a hierarchy of successive calibration steps that ensure each test reports a true quantity based on a certified reference material (CRM) from a national metrology laboratory. At the top of the hierarchy, metrologists calibrate a primary reference measurement procedure against the CRM. This primary procedure ultimately supports the calibration of a diagnostic test and controls.
Each step in this hierarchy introduces uncertainty. Consequently, according to the Consultative Committee for the Amount of Substance (CCQM) of the International Bureau of Weights and Measures (BIPM), the primary reference measurement procedure must have “the highest metrological properties.”1 It must operate according to a completed, described, and understood protocol, and it must have its uncertainty completely written down in terms of SI units.
“Any method that’s used as part of a primary reference measurement procedure, at its most accurate, would be SI traceable,” says Jim Huggett, PhD, principal scientist (nucleic acid research) in the LGC’s Health Science and Innovation Division, and senior lecturer in analytical microbiology at the University of Surrey. The calibration of any method should trace back to a universal standard, or CRM.
An SI-traceable reference measurement system for nucleic acids would benefit virtually all areas of research and medicine that rely on nucleic acid quantification. In medicine, perhaps the biggest long-term impact will be seen with the quantification of tumor load via circulating tumor DNA.
But for now, the focus of the world and the greatest need for absolute quantification concerns the COVID-19 pandemic. Accurate quantification of SARS-CoV-2 viral loads in patients helps authorities track the course of the disease and determine whether someone is contagious, with or without symptoms. In the absence of a reference standard, quantitative real-time PCR (qPCR) can produce only a qualitative result (that is, Ct values). Digital PCR, on the other hand, can fully quantify the virus in copies/µL.2
Variable qPCR standards
The current go-to method for quantifying primary reference materials is qPCR. However, qPCR does not offer absolute quantitation. To interpret a qPCR result in terms of nucleic acid quantity, users need to generate a standard curve using a serial dilution, a procedure that is prone to error and bias.
“RT-PCR has played a big role in helping us quantify molecular DNA and RNA, and it’s used extensively,” relates Huggett. “But in reality, it’s actually quite difficult to get a precise measurement and to know the trueness of the result. If you count 100 molecules, is it truly 100 molecules, or is it 50 or 5,000? That’s the type of variation you could see.”
In fact, digital PCR has already been used to quantify qPCR reference standards, exposing qPCR’s variability and inconsistency. This variability makes qPCR less sensitive to rare genetic variants associated with cancer. It also performs poorly when quantifying minimal residual disease, as seen with low circulating tumor DNA concentrations or viral load.
Another source of qPCR variability stems from differing amplification efficiencies of individual assays. Researchers must interpret the quantity of a target nucleic acid sequence in a sample based on sequence amplification and the subsequently generated fluorescent levels. This can be an unreliable process, with several factors that can interfere with the results.
For example, samples containing highly variable sequences or mismatches between primers or probe sequences, as well as secondary and tertiary nucleic acid structures, can all hamper the quantification of the target sequence. qPCR can also be impacted by inhibitors in the sample that reduce the overall level of amplification and affect the final quantification. For qPCR to be accurate, it must be calibrated using a standard curve, such as those produced using a primary reference measurement protocol that involves digital PCR.
Digital PCR as a primary reference measurement tool
Unlike qPCR, digital PCR does not require calibration; rather, it directly quantifies nucleic acid samples, making it eligible as an SI-traceable primary reference measurement procedure. Digital PCR works by directly counting the number of nucleic acids in a sample, bypassing the need for a standard curve.
The method uses a droplet reader to digitally count the number of target sequence copies in a sample that has been partitioned into tens of thousands of nanoliter-sized droplets. “Count” is a recognized dimensionless SI unit, which means digital PCR could potentially serve as an SI-traceable primary reference measurement procedure.
Over the past several years, metrology laboratories worldwide have demonstrated the accuracy and sensitivity of digital PCR in many areas of research, supporting its potential role as a primary reference measurement procedure. For example, David Dobnik, PhD, and his team at the National Institute of Biology, in Ljubljana, Slovenia, have even shown that dPCR can accurately and precisely quantify viral titers in plants3 and genetic modifications in foods.4
Along with quantifying primary reference standards, digital PCR is being explored as a tool for evaluating the accuracy and clinical utility of laboratory developed tests (LDTs). The National External Quality Assessment Service (NEQAS) in the U.K., for example, uses digital PCR to score LDTs from 1,500 testing laboratories around the world. Sandi Deans, PhD, a member of the U.K. National External Quality Assessment Service, believes the biggest impact digital PCR will have as a reference measurement tool in medical testing laboratories is in cancer.
“We do a lot of tumor testing, and tumor DNA tends to be present at low levels,” Deans explains. “We need to make sure we’ve got as much good-quality DNA present as possible to get a reportable result.” Digital PCR helps Deans and her colleagues at NEQAS determine if the samples being used for medical testing contain sufficient quality DNA to produce a true and actionable result. “Detecting these ctDNA molecules,” notes Deans, “means the difference between identifying sequence variants in tumors that can be treated using a personalized approach, or not.”
What accurate DNA quantification means for the future of medicine
At its core, a reliable primary reference measurement procedure is the key to many forms of measurement. In measurements of nucleic acids, accurate calibration ensures that life sciences researchers and physicians can achieve a result they can trust, publish, and use to make clinical decisions. With its ability to quantify nucleic acids without the need for calibration, digital PCR fits this need and will likely become an essential tool for laboratories around the world to calibrate their measurements against.
We cannot yet predict the full impact of digital PCR as a primary reference measurement tool or understand the extent to which the traceability of nucleic acid measurements will affect life sciences research, but it will undoubtably benefit clinical molecular laboratories and hospitals worldwide. To learn more about establishing digital PCR as an SI-traceable primary reference measurement tool, read “Counting DNA Molecule by Molecule,” a white paper from Bio-Rad Laboratories.5
Read more about the process of establishing digital PCR as an SI-traceable primary reference measurement tool in this whitepaper: www.bio-rad.com
1. Bunk DM. Reference Materials and Reference Measurement Procedures: An Overview from a National Metrology Institute. Clin. Biochem. Rev. 2007; 28(4): 131–137.
2. Liu X, Feng J, Zhang Q, et al. Analytical comparisons of SARS-COV-2 detection by qRT-PCR and ddPCR with multiple primer/probe sets. Emerg. Microbes Infect. 2020; 9(1): 1175–1179.
3. Mehle N, Dobnik D, Ravnikar M, Pompe Novak M. Validated reverse transcription droplet digital PCR serves as a higher order method for absolute quantification of Potato virus Y strains. Anal. Bioanal. Chem. 2018; 410(16): 3815–3825.
5. Corner AS. Counting DNA Molecule by Molecule.