Scientists from the University of Freiburg in Germany have developed a sequence-dependent universal detection technique for monitoring real-time PCR reactions that they say could significantly reduce the cost of performing real-time PCR.
The technique, called mediator probe PCR, or MP-PCR, makes use of label-free, sequence-specific primary probes combined with a complementary fluorogenic reporter probe, and is at least as sensitive and specific as hydrolysis probe PCR but with reduced cost due to the universal fluorogenic reporter, according to a recently published study.
The researchers have filed patents covering the technology and are now seeking corporate or other partners to help them commercialize the method as an inexpensive alternative to commonly used qPCR techniques.
"Basically the main cost in nucleic acid testing is the cost of the fluorogenic probes [such as] molecular beacons [and] hydrolysis probes," Bernd Faltin, a former PhD student in the University of Freiburg laboratory that developed the technique, told PCR Insider this week.
"If you have to test, for example, 10 different targets, then you have to order 10 different fluorogenic probes with high cost," he said. "Assuming that some probes are not that good — for example, at one specific locus you have to shift the sequence of nucleotides — then you'd have to re-order a new probe, and this will also increase expense."
Faltin and colleagues in the laboratory of University of Freiburg researcher Felix von Stetten attempted to tackle this issue by developing a more universal approach to real-time PCR detection.
"There were several universal sequence-dependent technologies described in the literature, so this was not completely new," Faltin said. "But we are the first group to describe a probe-based approach. The main motivation was to decrease the cost for oligonucleotides, but to combine this with high specificity."
The scientists described the MP-PCR technique in a paper currently in press in the journal Clinical Chemistry. The method first PCR amplifies target DNA using typical oligonucleotide primers and Thermus aquaticus polymerase. Then, the mediator probe, a sequence-specific bifunctional oligonucleotide, mediates real-time detection and is subsequently cleaved upon interacting with the target sequence.
This cleavage event then activates a second oligonucleotide, a fluorogenic universal reporter based on Förster resonance energy transfer, or FRET, with both cleavage and activation catalyzed by the polymerase, according to the paper.
During the course of MP-PCR, target amplification and detection take place simultaneously, and fluorescence emission from the FRET reaction accumulates with each successive amplification cycle, allowing real-time monitoring of the reaction using standard qPCR instrumentation.
In their study, the researchers compared their method to hydrolysis probe-based qPCR using TaqMan probes, which Faltin noted is the gold standard for real-time PCR monitoring. They used both techniques to amplify in real time human papillomavirus 18, Staphylococcus aureus, Escherichia coli, and human DNA dilution series.
Both techniques showed "exceptional linearity" in detecting DNA from the various dilution series. In addition, they tested the limits of detection when amplifying the HPV18 target DNA, finding that the mediator probe PCR could detect on average 78.3 DNA copies per 10-µL reaction while the TaqMan PCR could detect on average 85.1 copies per 10-µL reaction.
The scientists note in their paper that "the striking feature of our assay is the decoupling of amplification and fluorescence detection, which allows the use of standardized fluorogenic [universal reporter] oligonucleotides." As such, MP-PCR requires "only one single [universal reporter] layout that can be used for real-time detection of virtually any target DNA," meaning that this single reporter can be "synthesized in larger batches and at a lower price per unit than is possible for individual sequence-specific fluorogenic probes," they wrote.
Faltin added that a truly novel assay technique "has to be at least better, faster, or cheaper, and you're lucky if you have all of these things. We are definitely cheaper per reaction, and are at least comparable [in sensitivity and speed]. If you look at the limits of detection … the data are very reliable."
In their paper, the scientists presented a hypothetical scenario in which a cost assessment of an undisclosed international supplier revealed a cost of $245 per dual-labeled hydrolysis probe, $55 per mediator probe, and $600 per universal reporter. "Consequently, a set of eight individual hydrolysis probes would cost $1,960 … [while] a set of one universal reporter and eight mediator probes would be about $1,400," the researchers wrote.
Such cost savings could be important for any laboratory that performs large numbers of qPCR reactions, including laboratories involved in biomarker discovery or molecular diagnostic development.
"When I was getting my diploma we had no money for doing things like … real-time PCR," Faltin added. "For example, if a lab has 10 people and has to order 10 hydrolysis probes, but we have one standardized universal reporter in our laboratory, then of course we can benefit from [ordering in] bulk [and sharing]."
However, Faltin noted the cost savings of their method follows an "economy of scale."
"Cost is always relative … because you have individual discounts if you order TaqMan probes, or [other] hydrolysis probes, [and] you may pay a different price than I do. It depends on the lab and the discount," Faltin said.
Another attractive feature of MP-PCR is that it requires no different laboratory instrumentation or assay protocol, save for a slight adjustment to cycling times to account for necessary increased elongation times.
"People can amplify targets using the same protocol that is established in their laboratory," Faltin said. "They won't increase any annealing temperatures." The Freiburg group used a Qiagen Rotor-Gene Q for its experiments, but Faltin noted that if a researcher could, if he wanted to, alter extension time "to enable the assay for rapid cycling, with, for example, a Roche LightCycler."
Faltin said that the group has applied for patents on the method and is "very open" to identifying potential distributors for the MP-PCR technology. However, he is no longer involved with the project, having completed his doctoral degree and moved on to Bosch. He noted that Simon Wadle, a co-author on the paper and a current member of the von Stetten lab at the University of Freiburg, will be moving the project forward.
Von Stetten, who also co-authored the paper, wrote in an e-mail to PCR Insider that "one obvious scenario for commercialization of the mediator probe technology is that in the future, oligo synthesis companies will offer [it] in their product portfolio. The advantage for the customer would be cost reduction due to the large-scale synthesis of fluorogenic reporters. Oligo suppliers could offer a standard spectrum of fluorogenic reporters. Customers could order label-free mediator probes and add a low-cost reporter from the standard set."
He also noted that one stumbling block that could delay market entry is that there is currently "no specific mediator-probe and reporter design software available that could make oligo design easier. On the other hand, development of such software would be a market niche."