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Rice Team Develops Technique to Make Isothermal RPA Quantitative


NEW YORK (GenomeWeb) — To measure HIV viral load, researchers at Rice University in Houston have developed a technique that takes isothermal recombinase polymerase amplification, or RPA, to the next level.

The method, described as real-time, quantitative RPA, or qRPA, was published last month in Analytical Chemistry. It generates a single standard curve using internal control DNA, then uses an algorithm to analyze real-time fluorescence data and quantify HIV-1 DNA in samples.

The researchers showed that the qRPA technique was accurate over four orders of magnitude and was able to correctly predict an average HIV-1 DNA concentration within one order of magnitude. It was also most accurate for lower concentrations of template DNA, but still classed samples containing 10 copies or more of HIV-1 DNA as positive. All of these results suggest that their assay may meet the clinical benchmarks needed for a useful HIV-1 viral load test.

The group now hopes to develop this technology into a point-of-care assay to measure HIV viral load in low-resource settings.

In an interview with PCR Insider, co-authors Zachary Crannell and Brittany Rohrman explained that using the standard curve enables quantification of unknown samples, and, as such, one can then run a single sample without having to run a standard curve every time.

They also pointed out that a fast viral load assay could be very useful for HIV treatment. The turnaround time for testing dried blood spots in centralized testing labs can be up to four to six weeks, Rohrman said.

"A lot of people don't get treatment started as soon as they could, and a lot of people are lost to follow-up. [Our assay] enables people to be diagnosed at point of care, and get the treatment they need right away," she said.

The researchers, both doctoral students in the lab of Rebecca Richards-Kortum at Rice, calibrated the assay by amplifying an internal control derived from 200bp of Cryptosporidium parvum DNA.

This spiked-in control could have been any DNA not typically found in human blood, but the group had expertise with cryptosporidium, and published an RPA-based assay for it in Analytical Chemistry earlier this year.

In developing the control, Crannell said they worked with technical services staff at TwistDx, an Alere company.

There have been a number of recent reports of assays developed using the TwistDx RPA chemistry, as covered in PCR Insider. However, this is the first to make the RPA assay quantitative.

"People have shown that there is a correlation between when a signal turns positive and the amount [of nucleic acid] that's there," Rohrman said, "but nobody before had used a standard curve to quantify samples, and nobody had determined how accurate you could be when using this method for quantification."

Point-of-care HIV viral load assays are being pursued by other groups as well. A California Institute of Technology team is developing a digital PCR device called SlipChip running loop-mediated isothermal amplification assays to quantify HIV viral load, as reported in PCR Insider. And Alere announced last August that it intends to use the RPA chemistry acquired when it bought TwistDx to launch an HIV viral load assay.

The Rice team, meanwhile, is already in conversations with a few potential commercial partners, Crannell said. "Our lab always wants to try to get [molecular assays] into the hands of people that need them," he said.

"I think we're pretty set with our collaborators," Rohrman added, "but of course we're always open and happy to collaborate with others that might be interested." Crannell also noted that the group is developing other assays that might be amenable to collaboration.

The qRPA study was funded by The Bill and Melinda Gates Foundation, which has already provided $2.5 billion in HIV grants to organizations around the world, according to its website. The foundation has a special focus on developing point-of-care viral load testing for low-resource settings.

Crannell and Rohrman said they chose RPA from among other isothermal nucleic acid amplification technologies currently available — namely LAMP and nucleic acid sequence-based amplification (NASBA).

"NASBA and LAMP were things we had worked with in our lab, but we had some difficulties in getting them to work consistently," Rohrman said.

"One of the reasons [we] were drawn to [RPA] is that it works at a pretty low amplification temperature, and it is pretty simple to use and doesn't require too much infrastructure," Crannell said. "A lot of our technologies are built for low-resource settings, for use in labs that aren't necessarily as equipped as a diagnostic lab in the US or Canada."

The Analytical Chemistry study used a benchtop thermocycler to control temperature during the reaction. However, Crannell and Rohrman proposed that qRPA could be adapted to low-cost, commercially available readers, such as Qiagen's ESEQuant Tube Scanner or TwistDx's Twista portable real-time fluorometer, which has a heated incubation chamber powered by a rechargeable battery pack.

"We think one of the most novel things about this work is that it shows you can really quantify a single sample, instead of having to run a standard curve every time. We think that would be really valuable in the field," Rohrman said.

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