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Study Describes pH-based Method Potentially Useful for POC MDx

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NEW YORK (GenomeWeb) – Researchers at New England Biolabs have discovered that the release of protons associated with nucleic acid amplification is sufficient to drive a color change in pH-sensitive dyes under certain buffering conditions.

The new method was characterized using loop-mediated isothermal amplification (LAMP) and strand-displacement amplification (SDA) in a study published this week in the journal BioTechniques. Rapid, sensitive, and stable visual detection could enable lateral flow molecular tests, and the method is currently being developed by NEB for molecular diagnostics for parasitic infections, such as river blindness and elephantiasis, transmitted by insects.

The dyes used in the proof-of-principle study did not need to be modified in any way. "They're standard pH indicator dyes," Tom Evans, DNA Enzymes Division head at New England Biolabs, told GenomeWeb in an interview this week.

Instead, the advance of the research lay in proving that nucleic acid amplification reactions could be run successfully in minimal buffering conditions.

The release of hydrogen atoms is a well-known part of the chemical equation for DNA synthesis, Evans explained. "During extension of the DNA strand with a triphosphate mononucleotide, as part of the reaction a single proton is released for every dNTP added; if you look at the balanced  chemical equation for the polymerization reaction, that proton is in there," Evans said. "Most people just discount it, I guess."

The discovery was ultimately the result of challenging basic assumptions in the lab, Evans suggested. "We were actually playing around with different buffers and conditions, and one thing we tried was minimal buffer ... Sometimes you just try things," he said.

The "knee-jerk" in biology is to use buffers for chemical reactions, Evans explained. "You usually put in enough buffer that you know the pH isn't going to change," since a changing pH during a biochemical reaction is an unwanted variable, according to dogma. And if the pH doesn't change, pH indicator dyes are useless.

The group began by experimenting with the reaction parameters for nucleic acid amplification and found that minimal buffering conditions did not appear to affect the chemistry.

"We were surprised," Evans said. "We did the math, and [found that] with minimal buffers the amount of protons being released during amplification should, during the reaction, overpower the buffer." And yet, amplification still happened normally in these conditions.

Using a micro-pH meter, the group then detected significant pH changes during a number of standard amplification techniques. "We found a 2 to 3 pH unit change during, say, LAMP amplification, and with that knowledge we could then start to ask, 'how can we make use of this effect?,'" he said.

The group was initially concerned that the pH-sensitive dyes could themselves inhibit the reaction, but that was not the case.

Thus, the dye is ultimately "just a detection method" and can be used with almost any amplification technique, Evans said.

"We haven't tried recombinase polymerase amplification (RPA), but we have tried SDA, which is similar, and it works with that. We have used it with PCR, [and] it works really well with LAMP because [that method] produces so much DNA on amplification," he said.

Commercially available colorimetric pH-sensitive dyes also come in a wide range of colors and are easily detectable by the eye, Evans noted.

The development of molecular diagnostics for low-resource settings has been somewhat constrained by the read-out step of DNA and RNA amplification reactions. Fluorescence-based methods are certainly good to get digital, quantitative results, but might not be suitable for very low-resource settings. And, theoretically, a colorimetric technique in which antibody binds a fluorescent molecule adds an extra step compared to a pH-sensitive dye. 

A LAMP reaction for quantitative HIV detection using SlipChip technology reported in 2013 by researchers at Caltech, for example, requires a cell phone camera and a darkened shoebox to detect SYBR green fluorescence. Scientists at PATH are developing a disposable device for HIV detection that uses a lateral flow product – HybriDetect dipsticks from Milenia Biotec – but that method relies on antibody binding to a fluorescent molecule in order to create a readout that is visible to the naked eye. The HybriDetect dipstick method was also used in a recent study by a team in Germany that described an RPA-based assay for malaria parasite detection.

Although more work needs to be done, a colorimetric readout using a pH indicator dye could now enable one-step molecular lateral flow endpoint testing on par with home pregnancy tests, Evans said.

"New England Biolabs does a lot of exploratory applied and basic research," he said. "We don't always have a particular commercial outcome in mind." That being said, the firm has filed a patent on the method.

"We're not a diagnostics company," Evans noted, adding, "We're looking for partners, and based on what they're needs might be, we can discuss how we can help them meet those needs."

Meanwhile, NEB is working internally to explore using the method for diagnostic test kits, Evans said. His research group is collaborating with Clotilde Carlow of NEB's parasitology group to develop a test for insect vector surveillance that could also potentially be used for molecular diagnostics of neglected tropical diseases.

In the interview, Carlow said her group recently published an isothermal method to screen black flies for Onchocerca volvulus, the parasites that cause river blindness. That study did not include data on the pH dyes, but the dyes work well in that particular test, she noted. Her group is studying filariasis more generally, including elephantiasis, and she said the new method could potentially have applications for these projects as well as Lyme disease parasite detection.

"We need a very easy method that can be used in the field to go out and monitor the transmission in the insect population," she said. Molecular vector surveillance may be used to predict outbreaks and could be particularly critical in understanding the interplay between pharmaceutical treatment of humans and parasite levels in the insect host population, she said. Surveillance also could be used to determine the effects of mass drug distribution programs, for example, such as one in Africa being organized by Merck KGaA for schistosomiasis eradication. 

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