PCR | Digital PCR

Dr. Jim Huggett discovered first-hand the importance of standardization many years ago as a research fellow at University College London while working on diagnostics for developing countries. While identifying molecular markers of tuberculosis, he researched gene expression in patients from different populations who may have contracted the disease. When he measured the RNA in samples from Zambia and Tanzania, Huggett discovered that the findings differed between the two labs, a confounding result which led Huggett to ask an important question: “Was the discrepancy due to the variation between patient populations, or was it because the laboratories were performing the methods differently?”

At the time, Huggett’s lab was using quantitative PCR (qPCR) when the team realized they needed to develop a calibration solution to trust the results. “This opened my eyes to a whole field of science I had previously been unaware of: The science of measurement, of standardization, harmonization and measurement accuracy, otherwise known as metrology – a field to which I have dedicated much of my work over the last ten years,” says Huggett. Today, the use of molecular diagnostics is much more widespread and the methods employed have become more sophisticated. Still, the challenges remain the same: How can labs be sure to get the same result from a diagnostic test in Shanghai as one performed in London? Huggett believes digital PCR (dPCR) holds the answer.

An exact science

An exact science, digital PCR is a highly accurate approach for nucleic acid detection and quantification. While the basic principle is the same as other PCR technologies – it involves copying a DNA target of interest millions of times – it differs in that each DNA molecule is partitioned into individual PCR reactions and amplified separately. This means that it is possible to measure absolute numbers of DNA molecules, effectively counting them, something that is not possible with relative methods like qPCR. 

Huggett likes to use analog versus digital radio analogy to explain the key differences between qPCR and dPCR: With an analog radio, the dial must first be fine-tuned to get the desired station with the least interference. “Still, the quality depends on reception and the signal is subject to interference from static. This is qPCR. It is reliable but requires optimization to get a good result, and even then, you must contend with background noise. With digital radio, you simply call up the station and it is either there, with a clear signal, or not,” he says. The latter is like dPCR, which provides precise, binary results. It literally counts the presence or absence of DNA molecules. The clarity of results combined with a low error rate makes for an incredibly high level of precision. Digital PCR is well-suited to measure smaller quantitative differences.

A new level of patient care

Precision medicine, in which measurement of rare genetic variants is used to guide cancer therapy, is a great example of where this high level of precision can be useful. In a liquid biopsy, for instance, labs are interested in measuring tumor DNA that has made its way into the patient’s blood. In addition to tumor DNA, the liquid biopsy contains a lot of the patient’s normal genomic DNA. “Finding the tiny amount of tumor DNA in the large pool of normal DNA is like looking for a needle in a haystack. The sensitivity of dPCR makes this a perfect method for the detection of this tumor DNA from blood,” says Huggett. 

Most molecular oncology tests today look at the presence or absence of a tumor variant, but quantitative measures also provide value. By measuring levels of tumor DNA following cancer treatment, it could be possible to monitor patient response to a drug. Together with national measurement labs across the world, the lab at NML has been investigating the use of dPCR to quantitatively measure tumor DNA – and the results have been incredibly promising. “We demonstrated that dPCR can accurately count the number of DNA molecules in a given volume of liquid biopsy, with unprecedented agreement across different laboratories. This opens the door to a whole new level of cancer patient care,” says Huggett. 

The way forward

It also establishes dPCR as the first reference measurement procedure for quantitative DNA measurement, a fact that is incredibly exciting for Huggett. The lab has also used dPCR to quantify RNA molecules, for instance, comparing HIV RNAs to establish a standard for viral load testing. “We are now applying these methods to explore the international standardization of COVID-19 testing. Once again, we have been impressed with the results,” says Huggett. Other possible dPCR applications Huggett foresees are in measuring the efficiency of CRISPR alterations in DNA, or in evading the complications of amniocentesis by performing NIPT dPCR assays.

Dr. Huggett is aware that this potential is accompanied by a variety of challenges. He and his lab are working hard to address such topics such as how to ensure that sample purification methods are standardized, and what thresholds need to be set for data analysis. There is also a need for simpler, more affordable instruments to enable labs around the world to harness the power of dPCR technology. “But the future is promising, and I can see a day when every lab will have a dPCR instrument and be able to perform highly reproducible quantitative measurements. And perhaps one day we can truly be sure that a diagnostic test result in Shanghai is the same as one achieved in London,” says Huggett.