As an indispensable means of diagnosing COVID-19, PCR has speed, sensitivity and reliability, and can be used in a wide range of clinical applications
Due to the COVID-19 pandemic, polymerase chain reaction (PCR) testing has become routine, and more than one million PCR tests are performed every day in the United States alone. The technology now commonly referred to as RT-PCR detection has become the key to the global response to the pandemic.
Although everyone says "RT-PCR", the term can be confusing. "RT" can stand for "real time" or "reverse transcription". In addition, RT-PCR testing may or may not include a technique called quantitative PCR (qPCR). Finally, qPCR sometimes has the same meaning as real-time PCR.
For clarity, the gold standard diagnostic test for COVID-19 should be called "qRT-PCR" to emphasize that it combines reverse transcription PCR and quantitative PCR. Like other PCR techniques, qRT-PCR relies on PCR, which is a common method for making copies of many small DNA fragments. In addition, qRT-PCR uses reverse transcriptase to create complementary copies of RNA in the sample. Finally, qRT-PCR uses qPCR, which monitors the amplification of target DNA molecules during PCR by measuring the fluorescent signal from the binding of fluorescent dyes or probes. (In qPCR, fluorescence is monitored in real time, that is, during the amplification process, not at the end of the amplification as in traditional PCR.)
Because qRT-PCR combines PCR amplification, reverse transcription, and fluorescence monitoring, it can measure the number of RNA molecules that have been targeted for analysis, even those that are too few to be measured. Needless to say, these RNA molecules may correspond to viral RNA, such as RNA from the SARS-CoV-2 virus.
Of course, the clinical application of PCR technology is not limited to COVID-19 testing, but also research applications. For example, reverse transcription can be used to monitor gene expression or mRNA synthesis. If qPCR uses fluorescent DNA probes instead of dyes, it can measure multiple DNA targets—that is, it can achieve multiple applications. Another PCR technique is digital PCR. It involves dividing the PCR sample into thousands of nanodroplets and performing a separate PCR reaction on each nanodroplet. In digital PCR, "number" refers to the absolute quantification of the target nucleic acid. Digital PCR does not rely on references or standards to derive absolute quantities from relative or "analog" measurements. Therefore, it can have higher accuracy.
The 8th qPCR and Digital PCR Conference to be held in London from December 6th to 7th will discuss the latest developments in qPCR and digital PCR. The presentations at this event will focus on the challenges of using qPCR and digital PCR in clinical settings-challenges such as accuracy, reproducibility, assay optimization, multiplexing, and standardization. This article discusses several of these challenges and shares insights from the most interesting speakers at the upcoming event.
The pandemic highlights some of the shortcomings of PCR testing. "Obviously, testing is an important part of the response, but PCR testing is an extremely time-consuming infrastructure," said Dr. Stephen Bustin, professor of molecular medicine at Anglia Ruskin University. If you are lucky, he went on to say that it may take you six hours to get a sample, but it usually takes two to three days to get an analyzable result.
Bustin has been working in PCR for decades. In 2007, he became famous as an expert witness for the US Department of Justice. In this capacity, he re-analyzed the RT-qPCR data, which supports Andrew Wakefield's notorious work linking MMR vaccines to autism.
Through his work, Busin learned that PCR often performs poorly and is improperly applied. He also found that these problems have an impact on real life. In the case of Wakefield's reanalysis, he found a contamination problem in the original PCR analysis of intestinal samples from children with autism.
Bustin has been committed to making PCR analysis faster and more reliable. He recalled his reaction after a colleague reported that extreme PCR could perform 30 amplification cycles in 20 seconds. Bustin realized that the speed of extreme PCR could be valuable in COVID-19 diagnosis. He still has this feeling: "We need a personalized bedside system where you can perform PCR in the shortest time possible."
Since 2019, Bustin has been working on a method of running 30 PCR amplification cycles in 75 seconds. At the conference, he plans to explain how to sample, enrich the RNA content, perform a reverse transcriptase reaction, and then perform rapid PCR on the DNA.
He explained that traditional PCR relies on a heating block to adjust the temperature, while his method uses a robot to quickly move the sample between hot water baths. "I can't tell you much about it," he said. "Universities are very interested in it, there are many patents."
Although Bustin must be discrete, he does work with OptiSense, a small analytical instrument company based in Horsham, UK. The collaborators developed a two-year business plan to achieve the miniaturization of the technology. They are currently developing prototype instruments.
"If it works, it will completely change the way we do PCR," Busin asserted. "The goal is to perform PCR in five minutes. Therefore, you may go to Boots [UK pharmacist] for SARS-CoV-2 or flu testing. You can perform the test before entering the waiting room or hospital environment. Visitors can enter Nursing homes are tested before, or people can be tested at airports or on cruise ships-the potential uses are limitless."
The Cell and Gene Therapy Catapult (CGTC) in the United Kingdom has been working with industry partners and academic collaborators to advance the application of PCR in gene therapy manufacturing. This is according to Dr. Lily Li, a senior scientist in CGTC viral vector analysis. Li will present a study at the conference that compares qPCR with droplet digital PCR (ddPCR) to monitor the copy number of genomic viral vectors in the manufacture of adeno-associated virus (AAV).
"When manufacturers make these AAV gene therapy products, they need to monitor the AAV genome copy number," Li said. "One of the most commonly used methods is through qPCR, which is why adequate AAV characterization is essential for process development, manufacturing, clinical dosage, and final product safety."
Li pointed out that the accuracy of qPCR may be poor for AAV characterization, and when applied to complex gene therapy products, qPCR may perform poorly.
"All these complexities may affect how qPCR (one of the most common [PCR] methods) works," she explained. Her research shows that the performance of ddPCR is better than qPCR. "It is less susceptible to complex factors, and it is more powerful," she explained in detail. "We know it is more capable of handling these variables." However, she admits that qPCR is cheaper than ddPCR and has higher throughput, and that many manufacturers still consider qPCR to be the gold standard technology.
Dr. Mikael Kubista, head of gene expression profiling at the Institute of Biotechnology of the Czech Academy of Sciences, said that the latest PCR innovation that is expected to advance accurate diagnosis is two-tailed PCR. "I think we published it for the first time three or four years ago," he recalled. "That is to publish a microRNA test application."
Conventional qPCR uses two primers, preferably one probe. Primers are short fragments of single-stranded DNA, located on either side of the DNA region to be copied (and amplified). At the same time, the probe is a fluorescently labeled DNA oligonucleotide, which binds to the downstream of the primer and emits fluorescence when the DNA is cleaved during the amplification process. According to Kubista, primers and probes are often 20-25 bases in length, so DNA/RNA molecules shorter than 50 bases cannot be detected or amplified.
"In general, this has been a major issue when analyzing short molecular targets like microRNA," he said. "If you use fragmented materials, the standard method is to make the RNA longer to fit two primers, but if you make the original RNA target longer, you must include another reaction, which is extension."
Kubista explained that lengthening the original RNA would add additional process steps, which would affect PCR yield. He explained that you will also lose specificity because the primer is aimed at a small, specific base sequence.
He explained that two-tailed PCR overcomes this problem by using a single molecule that hybridizes to both ends of the microRNA to trigger. Although each half-probe itself is too small to form a stable interaction with microRNA, when they are placed on the same molecule, the hybridization efficiency of the half-probe is the same as that of a conventional primer.
"You gain sensitivity because you don't have to include additional elongation response," Kubista explains. "You perform PCR directly on the target, and you also have excellent specificity." He said that two-tail PCR can detect as few as one molecule of sequence variation between 100 and 1,000 sequences in a drop of digital PCR.
After the two-tail PCR technology first appeared in published works in 2017, BioVendor began to commercialize it, and the company currently provides the technology in an off-the-shelf test format. The first panel to detect SARS-CoV-2 microRNA was developed using two-tailed PCR. The technology can also be used to monitor organ rejection. In these applications, the technology can monitor donor DNA—that is, DNA from the donor’s heart, lung, kidney, or liver—that enters the patient’s blood during rejection. Kubista claims that tiny fragments of donor DNA can be detected by two-tailed PCR.
Another researcher dedicated to improving the specificity of DNA detection in patients’ blood is Dr. Viktor A. Adalsteinsson, associate director of the Getzner Cancer Diagnostic Center at the Broad Institute of MIT and Harvard University. Adalsteinsson focuses on improving the sensitivity of detection of minimal residual disease (MRD) technology.
"People are very interested in tracking MRD (cancer left after treatment)," he said. "There are millions of cancer patients undergoing surgery for early-stage cancer. However, if it is not known whether MRD is present in other parts of the body, it is difficult to assess whether further treatment is needed or the risk of future recurrence.
"When there is very little tumor DNA in the blood, it is very unlikely that all the mutations in the patient's tumor will be drawn into any one of the blood. We believe that finding all the mutations in the patient's tumor genome can increase the detection rate."
He will present a study at the conference that shows that tracking more mutations in each patient can increase the likelihood of detecting MRD. The study describes how Adalsteinsson and colleagues used the ultra-sensitive blood test they developed for cell-free DNA to track mutations. The test uses exome sequencing for patient-specific single nucleotide variations.
The researchers compared their test results with the results of the ddPCR test. Both tests are used to evaluate a group of breast cancer patients. The researchers found that the error rate of the new test was a thousand times lower.
Adalsteinsson and colleagues developed several other new methods. For example, they developed a tandem original duplex (CODEC) for error correction, a sequencing method that combines the massively parallel nature of next-generation sequencing with the single-molecule capabilities of third-generation sequencing. They developed Duplex-Repair, a method that can limit the re-synthesis of internal double-stranded base pairs, rescue the effects of induced DNA damage, and provide more accurate double-stranded sequencing. They developed secondary allele enrichment sequencing by identifying oligonucleotides (MAESTRO), which is a method of enriching mutations. According to Adalsteinsson, MAESTRO makes it possible to track genome-wide tumor mutations in the blood.
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