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Study Finds Digital PCR No Better than Real-Time PCR for Low-Level HIV Detection, Quantification

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Droplet digital PCR is no better than real-time PCR at detecting and quantifying low levels of cell-associated HIV-1 genetic material from samples taken from patients both on and off antiviral therapy, according to newly published study.

In addition, digital PCR analyses take more time and may be more expensive than real-time PCR for this specific application, said one of the study's authors, who nonetheless also noted that he is "optimistic" about the digital PCR platform — Bio-Rad's QX100 Droplet Digital PCR system — for future similar HIV studies.

However, according to Bio-Rad scientists, the results of the study may have been due to sub-optimal assay conditions and not using the digital PCR platform to the fullest extent of its capabilities. In addition, Bio-Rad noted that other research groups have already found the platform to be significantly more sensitive than qPCR for detecting low levels of HIV DNA.

In the study, published in the December 2012 issue of Journal of Virological Methods, scientists from Brigham and Women's Hospital and Harvard Medical School compared the ability of Bio-Rad's QX100 system with previously validated TaqMan-based real-time PCR assays to detect and quantify total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy.

"We were interested in buying the machine, partly because this was an opportunity to look at a platform with potentially better sensitivity for very low minority copies, which is what we're interested in with HIV curative and reservoir research," said Timothy Henrich, corresponding author on the paper and an infectious disease researcher at Brigham and Women's and Harvard.

"We thought this was a promising technology and that we'd give it an initial go-through to see how well it performs compared to standard techniques that we currently use," he added. "It's a significant investment … so we wanted to give it a test spin."

Henrich noted that his research team was aware that other HIV researchers were also interested in the QX100 because of its purported high level of sensitivity and ability to quantitate low DNA copy numbers, a crucial aspect of HIV research.

"The main purpose we wanted to use it for was to see how it would probe cellular reservoirs for HIV, because they can exist in very low numbers — basically one in a million resting CD4 cells can harbor HIV," he said. "And we wanted to see what this could do in terms of sensitivity, as well as reproducibility and precision."

To compare the dynamic range of the two methods, Henrich and colleagues used them both on serial dilutions of HIV-1 DNA and 2-LTR circles. In addition, they obtained 5 million peripheral blood mononuclear cells from three HIV-1 infected viremic patients and four patients on suppressive antiretroviral therapy, then extracted and purified DNA from these cells and quantified HIV-1 DNA and 2-LTR circles using either the real-time PCR assays or ddPCR on the Bio-Rad system.

The researchers found that expected copy numbers measured by ddPCR were comparable to standard input values with the exception of the HIV-1 DNA amplicon standard, where ddPCR detected approximately 60 percent fewer HIV-1 DNA copies than expected. However, they also found that ddPCR and real-time PCR had similar sensitivities, as both obtained positive signals in two of three wells for the three-copy standard.

When comparing the two methods for measuring HIV-1 DNA and the human CCR5 gene in cells from the infected patients, the researchers found that HIV-1 DNA from patient samples both on and off therapy ranged from 27 to 4,894 copies per million cells using real-time PCR; and ranged from 23 to 2,289 copies per million cells using ddPCR. Overall, ddPCR enumerated approximately 10 percent to 60 percent fewer DNA copies compared with real-time PCR, but demonstrated similar detection sensitivity, the researchers wrote in their paper.

"It's a new technology, and it may be more sensitive [than real-time PCR]," Henrich said. "I don't discount that, and we're still interested in doing more with this technology. We need to do more rigorous studies on more samples, at lower levels, just to see what the reproducibility is. But we were struck with how similar it was to real-time PCR using TaqMan probes. When we did dilutional samples to see where our threshold of detection was, we were able to get a similar threshold using the same method with both platforms."

"That said, the machine is precise [and] very reproducible — probably more so than real-time PCR," he added. "At the same time, the overall results we were getting were very similar, which means that the results that we had with the assay that we ran — and of course we are always up for improving the assay for the ddPCR machine — [were] fairly similar to real-time PCR."

According to Bio-Rad, the majority of its QX100 customers thus far have achieved a lower limit of detection and quantitation with droplet digital PCR than with real-time PCR, primarily because of the former's ability to partition samples into hundreds of thousands of individual reaction volumes.

In particular, Bio-Rad representatives cited a recent study conducted by researchers at biotechnology firm Sangamo, who plan to use the QX100 to measure HIV DNA levels in patients receiving an experimental zinc finger DNA-binding protein-based technology being developed by the company to potentially induce cellular resistance to HIV without the use of drugs.

So far, Sangamo researchers have found that at low copy numbers in two separate assays, real-time PCR has shown a ten-fold difference in target DNA concentration, making it difficult to determine whether the data was a result of experimental variation or biologically real. With ddPCR technology, the difference was only two-fold.

"The QX100 allowed us to easily, accurately, and reproducibly measure low copy events in genomic DNA," Gary Lee, senior scientist at Sangamo BioSciences, said in a statement provided by Bio-Rad. "ddPCR technology gave us confidence in our results, which is critical in advancing any program. We have been able to demonstrate that the QX100 is the technology of choice when it comes to rare event detection applications, particularly for clinical studies."

More information about Sangamo's work can be found on Bio-Rad's website, and Sangamo recently published initial results of its work with the QX100 in a poster presented last month at the annual Interscience Conference on Antimicrobial Agents and Chemotherapy in San Francisco.

According to Richard Kurtz, marketing manager for amplification at Bio-Rad, limit of detection and sensitivity "depends a little bit on your assay design and assay optimization. It kind of depends on how you want to define sensitivity and what you're looking for when you're doing these kinds of comparative analyses … and maybe taking the time to really push the optimization steps for digital PCR versus a real-time PCR assay."

George Karlin-Neumann, senior director of ddPCR assay development in Bio-Rad's Digital Biology Center, added that the Sangamo research "really tried to push the sensitivity, and part of doing that means you're really loading a lot more DNA than perhaps [the Brigham and Women's and Harvard] researchers. [Sangamo] have data where they push the system, and it's not clear to me whether they pushed it to that same regime [in the JVM study]. It may be that if you're not using a demanding regime, then maybe you get a comparable result between [ddPCR and real-time PCR], but I think the higher sensitivity … is in some of these more demanding situations."

The authors of the JVM paper also highlighted some other shortcomings of ddPCR in their hands. Some of these shortcomings were highlighted in the paper — for instance, the need to digest extracted genomic DNA prior to ddPCR experiments "because DNA strands longer than several hundred base pairs do not package efficiently into the picoliter droplets," they wrote.

In addition, Henrich told PCR Insider that although his group did not do an intensive cost-benefit analysis comparing ddPCR to real-time PCR, he believed that ddPCR "isn't much cheaper" and "takes longer to do" than real-time PCR, in part because of the multiple steps involved in digital PCR and the need to pre-digest large DNA strands.

Regarding not being able to package DNA into picoliter droplets, Bio-Rad's Karlin-Neumann said that such an assertion was "patently untrue," and that Bio-Rad researchers have been able to "take DNA of many [kilobases] or tens of [kilobases], and they will package fine. It may be useful to reduce the size of DNA because of a secondary structure that may occur that makes the DNA target less readily accessible, but that would be true of qPCR, as well."

In addition, Bio-Rad's Kurtz conceded that ddPCR may indeed cost more than real-time PCR depending on the experiment, but that the richness in data produced by the former is invaluable.

"On the overall cost, it depends on how you want to spin it," Kurtz said. "We have been pretty clear in stating that our price per reaction in running a digital PCR experiment is about $3. And the price you're going to spend for real-time PCR varies on how much you spend on your master mix, and the overall volume that you're running for your reactions."

Competing digital PCR vendors such as RainDance Technologies have claimed that droplet digital PCR is cheaper per data point, and that their digital PCR platform is in turn the most cost-effective because it can produce the largest number of individual droplet-based reaction volumes.

"If you want to spin it as getting more data points for less money, that's true, because … [if] we have an analysis of say 15,000 droplets, and each one of those 15,000 is a data point, then if you take that $3 and divide it by 15,000, it doesn't cost you a lot of money per data point," Kurtz said.

"That's sort of the spin that one of our competitors is pushing out there," he added. "But in reality, it's cost per answer, or cost per sample, and we sort of push that … in reality it is a little more expensive to do this; you have to invest in the partitioning of your samples into the droplets, and that does push up the price a little bit."

Despite the shortcomings that Henrich and colleagues perceived for droplet digital PCR, he said that he is "optimistic" that the technology will soon begin to replace other methods, and that it just needs refinement before it can be dependably used for detecting low-level viral targets in infectious disease research.

"We're considering moving forward with [the QX100]," Henrich said. "At this point [we're not sure] exactly what we'll be doing with it … but we're still very interested in seeing what it can do. The theoretical benefits versus the benefits that have been shown … [have] all been corporate-sponsored research … so take that with a grain of salt. It's been shown to be better in particular experiments, but is that going to be extrapolatable to everything that we do? I think that is still an open question, for me."

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