Polymerase chain reaction (PCR) tests have become routine because of the COVID-19 pandemic, with more than a million PCR tests being carried out daily in the United States alone. What are now commonly known as RT-PCR tests have become key to the world’s pandemic response.

Although “RT-PCR” is on everyone’s lips, the term can be confusing. The “RT” can stand for “real time” or “reverse transcription.” Also, RT-PCR tests may or may not incorporate a technique called quantitative PCR (qPCR). Finally, qPCR is sometimes taken to mean the same thing as real-time PCR.

For the sake of clarity, the gold standard diagnostic test for COVID-19 should be called “qRT-PCR” to emphasize that it incorporates reverse transcription PCR and quantitative PCR. Like other PCR techniques, qRT-PCR relies on PCR, a common method for making many copies of small DNA segments. In addition, qRT-PCR employs a reverse transcriptase, which creates complementary DNA copies of the RNA in a sample. Finally, qRT-PCR uses qPCR, which monitors the amplification of a targeted DNA molecule during the PCR by measuring fluorescent signals from the binding of fluorescent dyes or probes. (In qPCR, fluorescence is monitored in real time, that is, during amplification, not at the end of amplification, as in conventional PCR.)

Because qRT-PCR combines PCR amplification, reverse transcription, and fluorescence monitoring, it is able to measure the amount of RNA molecules that have been targeted for analysis, even RNA molecules that are present in quantities that would otherwise be too small to measure. Needless to say, these RNA molecules may correspond to viral RNA, such as the RNA from the SARS-CoV-2 virus.

Of course, PCR technologies have clinical applications beyond COVID-19 testing, as well as research applications. For example, reverse transcription can be used to monitor gene expression or mRNA synthesis. And if qPCR uses fluorescent DNA probes rather than dyes, it can measure multiple DNA targets—that is, it can realize multiplexing applications. Yet another PCR technology is digital PCR. It involves partitioning PCR samples into thousands of nanodroplets, with a separate PCR reaction performed on each one. In digital PCR, the “digital” refers to the absolute quantification of target nucleic acids. Digital PCR does not rely on references or standards to derive absolute quantities from relative or “analog” measurements. Accordingly, it is capable of greater precision.

The latest advances in qPCR and digital PCR will be discussed at the 8th qPCR & Digital PCR Congress, which is to be held December 6–7 in London. The event’s presentations will focus on the challenges of using qPCR and digital PCR in clinical settings—challenges such as accuracy, reproducibility, assay optimization, multiplexing, and standardization. Several of these challenges are discussed in this article, which shares insights from the upcoming event’s most intriguing speakers.

Making PCR faster

The pandemic has highlighted some of the deficiencies in PCR testing. “It’s become obvious testing is a crucial part of the response, but PCR testing is extremely infrastructure- and time-consuming,” says Stephen Bustin, PhD, professor of molecular medicine, Anglia Ruskin University. If you’re lucky, he continues, it might be six hours before you have a sample, but it’s usually two or three days until you have an analyzable result.

Speeding up the workflow
At Anglia Ruskin University, Stephen Bustin, PhD, has been working to make PCR analysis faster and more reliable. He believes that with the development of standardized workflows and optimized protocols, RT-qPCR systems could be devised that would provide five-minute reporting/recording of test results at the point of care.

Bustin has worked with PCR for decades. He achieved a measure of fame in 2007 as an expert witness for the U.S. Department of Justice. In this capacity, he presented a reanalysis of the RT-qPCR data underpinning Andrew Wakefield’s infamous work linking the MMR vaccine with autism.

Through his work, Bustin has learned that PCR is often performed poorly and applied inappropriately. He has also found that these problems have real-life consequences. In the case of the Wakefield reanalysis, he uncovered contamination issues in the original PCR analysis of intestinal samples taken from autistic children.

Bustin has been working to make PCR analysis faster and more reliable. He recalls how he reacted after a colleague reported that extreme PCR could perform 30 cycles of amplification in 20 seconds. Extreme PCR’s speed, Bustin realized, could be valuable in COVID-19 diagnostics. He still feels that way: “We need a personalized point-of-care system where you can do PCR in as little time as possible.”

Since 2019, Bustin has been working on a method to run 30 cycles of PCR amplification in 75 seconds. At the congress, he plans to explain how to take a sample, enrich the RNA content, perform a reverse transcriptase reaction, and then run a rapid PCR on the DNA.

He explains that conventional PCR relies on a heating block to adjust temperature, whereas his method uses a robot to move samples rapidly between heated water baths. “I can’t tell you a lot about it,” he says. “The university is quite interested in it, and there are a lot of patents.”

Although Bustin must be discrete, he does share that he’s working with OptiSense, a small analytical instrument company based in Horsham, U.K. The collaborators have a two-year business plan to miniaturize the technology. They are currently developing a prototype instrument.

“If it works, it’s going to revolutionize how we do PCR,” Bustin asserts. “The aim is to do a PCR in five minutes. So, you might pop down to Boots [a U.K. pharmacist] and get a SARS-CoV-2 or flu test. You could test before you go into a waiting room or a hospital setting. Visitors could get tested before entering a care home, or people could get tested in an airport or on a cruise—the potential uses are quite limitless.”

Amplifying the applications

The U.K.’s Cell and Gene Therapy Catapult (CGTC) has been working with industry partners and academic collaborators to progress the use of PCR for gene therapy manufacturing. That’s according to Lily Li, PhD, a viral vector analytical senior scientist at the CGTC. Li will be presenting a study at the convention that compares qPCR with droplet digital PCR (ddPCR) for monitoring genome viral vector copy number in adeno-associated virus (AAV) manufacturing.

“When manufacturers make these AAV gene therapy products, they need to monitor the genome copy number of the AAV,” Li says. “One of the most-used ways is by qPCR, and that’s why adequate AAV characterization is critical for process development, manufacturing, clinical dosing, and ultimately product safety.”

Li notes that the precision of qPCR can be poor for AAV characterization, and that qPCR can behave suboptimally when applied to complex gene therapy products.

“All this complexity may affect how qPCR, one of the most common [PCR] methods, works,” she explains. Her research indicates that ddPCR performs better than qPCR. “It’s less susceptible to complex factors and more robust,” she details. “We know it’s more capable of processing these variables.” She acknowledges, however, that qPCR is cheaper and delivers higher throughput than ddPCR, and that many manufacturers still consider qPCR to be a gold standard technique.

Improving PCR precision

A recent innovation in PCR that promises to advance precision diagnostics is two-tailed PCR, says Mikael Kubista, PhD, head of gene expression profiling, Institute of Biotechnology, Czech Academy of Sciences. “I think we first published on it three or four years ago,” he recalls. “And that was the publication of an application for microRNA detection.”

two-tailed RT-qPCR illustration
An advanced PCR technology called two-tailed RT-qPCR is being developed for microRNA detection by Mikael Kubista, PhD, head of gene expression profiling, Institute of Biotechnology, Czech Academy of Sciences. Instead of using a single probe, two-tailed PCR uses two hemiprobes that bind to different stretches of targeted microRNA and are connected by a tether. Using two probes ensures high sensitivity and enables discrimination of highly homologous microRNAs.

Regular qPCR uses two primers and, optimally, a probe. The primers are short sections of single-stranded DNA that flank the region of DNA to be copied (and amplified). The probe, meanwhile, is the fluorescently labeled DNA oligonucleotide that binds downstream of the primer and fluoresces when the DNA is cut during amplification. According to Kubista, the primers and probe tend to be 20–25 bases long and are thus unable to detect or amplify a DNA/RNA molecule shorter than 50 bases.

“Generally, this has been a major problem when analyzing short molecular targets like microRNAs,” he says. “If you work with fragmented material, the standard method is to make the RNA longer to fit the two primers, but if you make the original RNA target longer, you have to include another reaction, the elongation.”

Kubista explains that elongating the original RNA compromises the PCR yield by adding an additional process step. You also lose specificity, he explains, because the primer targets a small, specific sequence of bases.

Two-tailed PCR overcomes this problem by priming with a single molecule that hybridizes to both ends of the microRNA, he explains. Although each hemiprobe is too small on its own to form a stable interaction with the microRNA, the hemiprobes hybridize with the same efficiency as a regular primer when they are put together on the same molecule.

“You gain sensitivity because you don’t have to include an additional elongation reaction,” Kubista explains. “You PCR the target directly, and you also have fantastic specificity.” Two-tailed PCR, he says, can detect as little as one molecule of sequence variation among 100 to 1,000 sequences in a digital PCR droplet.

After the two-tailed PCR technology first appeared in published work in 2017, it began to be commercialized by BioVendor, which currently offers the technology in the form of off-the-shelf assays. The first panel to detect SARS-CoV-2 microRNAs was developed using two-tailed PCR. The technology also has applications for monitoring organ rejection. In these applications, the technology can monitor donor DNA—that is, DNA from a donor heart, lung, kidney, or liver—that enters the patient’s bloodstream during rejection. The tiny fragments of donor DNA, Kubista asserts, are detectable with two-tailed PCR.

Turning to advanced sequencing techniques

Another researcher working to improve the specificity of DNA detection in patient’s blood is Viktor A. Adalsteinsson, PhD, associate director of the Gertsner Center for Cancer Diagnostics at the Broad Institute of MIT and Harvard. Adalsteinsson is focused on improving the sensitivity of techniques for detecting minimal residual disease (MRD).

“There’s been a lot of interest in tracking MRD—the cancer left after treatment,” he says. “There are millions of cancer patients that undergo surgery for an early-stage cancer. But if there’s no way to know if there’s MRD left elsewhere in the body, it’s difficult to assess the need for further treatment or the risk of a future recurrence.

“When there’s very little tumor DNA in the blood, the likelihood that all mutations from a patient’s tumor are drawn in any one tube of blood is slim. We think that looking for all mutations in a patient’s tumor genome can improve detection.”

He will be presenting a study at the congress showing that tracking more mutations per patient improved the likelihood of detecting MRD. The study describes how Adalsteinsson and colleagues tracked mutations using an ultrasensitive blood test they developed for cell-free DNA. The test used exome sequencing for patient-specific single-nucleotide variants.

The researchers compared the results from their test to the results from a ddPCR test. Both tests were used to evaluate a cohort of breast cancer patients. The new test, the researchers found, had a thousand-fold lower error rate.

Adalsteinsson and colleagues have developed several other new methods. For example, they developed Concatenating Original Duplex for Error Correction (CODEC), a sequencing method that combines the massively parallel nature of next-generation sequencing with the single-molecule capability of third-generation sequencing. They developed Duplex-Repair, a method that can limit interior duplex base pair resynthesis, rescue the impact of induced DNA damage, and afford more accurate duplex sequencing. And they developed Minor Allele Enriched Sequencing through Recognition Oligonucleotides (MAESTRO), a method for mutation enrichment. According to Adalsteinsson, MAESTRO has made it possible to track genome-wide tumor mutations in blood.

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