NEW YORK – The ideal infectious disease diagnostic test would involve minimal sample preparation, high sensitivity and specificity in detecting a pathogen early in infection, with results available quickly and at a low cost.
For the dengue virus — and potentially other targets as well — a new test method may now be able to meet those requirements. Researchers have developed a technique using DNA nanostructures that can detect intact dengue virus from whole blood in about two minutes, on the first day of infection, for a cost of $.15 per test. The team plans to further develop the technology to detect blood stream infections and circulating cancer cells.
The team was comprised of researchers from Rensselaer Polytechnic Institute, Nanjing University of Posts and Telecommunications, New York State Department of Health's Wadsworth Center, and New York University. Their technology was described last month in Nature Chemistry.
The method relies in part on the fact that the surface of many pathogens is studded with signature proteins laid out in stereotyped patterns. Dengue, for example, has a protein called envelop protein domain 3, or ED3, arranged on its surface in precise star-shaped clusters.
Led by Xing Wang, a researcher formerly with Rensselaer Polytechnic Institute now at University of Illinois at Urbana-Champaign, the team designed a star-shaped DNA nanostructure that precisely positioned aptamers targeting the ED3s. Each five-pointed star-shaped structure has 10 vertices, and each vertex got an aptamer.
"When you do this pattern-matching polyvalence strategy, you are able to increase the binding strength dramatically," Wang explained in an interview.
A five-pointed star can also be broken down into a pentagon with five triangle arms, so the team also proved that attaching aptamers to triangle-shaped DNA nanostructures was not as sensitive as the star shape. Using 10 aptamers attached to the star-shaped DNA nanostructure, the binding affinity was enhanced by a thousandfold as compared to just using the aptamers as monomers, Wang said.
In the pentagon center of the star, the team placed stem-loop motifs with fluorophores and quenchers attached. When the DNA star aptamers bind to their targets, this stretches apart the stem and loop, unquenching the fluorophore and resulting in a signal that can be detected with a simple fluoroscope.
The researchers did not test patient samples, but they did look at dengue virus spiked into human serum and plasma, yielding a limit of detection of 100 pfu/ml and 1,000 pfu/ml, respectively.
Gold standard diagnostics for dengue virus typically involve either direct detection of viral RNA or detection of the patient's immune response to infection.
For the former, RT-qPCR is used, requiring sample preparation steps and, usually, skilled technicians and expensive instrumentation, although it can often detect infection on the day of symptom onset.
For the latter, ELISA is often performed to detect antibodies to virus or a protein on the surface of the virus called non-structural protein 1, or NS1. But this method also usually needs some skill to perform, and serology testing won't yield a positive result until about seven days after the initial infection.
Although PCR-based methods use amplification to enhance detection, the extraction and reverse transcription required for all RNA viruses can lead to loss of viral particles, Wang said. The team also compared its method to the most sensitive of the gold standard methods and found that RT-qPCR had a limit of detection of 1,000 pfu/ml, and a turnaround time of four hours.
Wang explained that the RT-qPCR test is considered to be "direct sensing" of the virus since it is detecting RNA directly from the virus. The DNA nanostructure method was superior to PCR, however, and because it detects intact, active virus, he deemed the method (direct)2 sensing.
"Our (direct)2 sensing method can catch all virus particles, without losing any signal," he noted. Indeed, the team estimates it can detect dengue virus in blood even before symptoms of infection start, when viral concentrations are still extremely low.
The DNA nanostructures are not new, of course, but Wang said using them for pattern recognition-enabled diagnosis is novel.
Another common method, using DNA origami, could be used for bigger targets like bacterial cells or cancer cells, but viruses are so small that the nanostructures were preferable, he said.
Synthesizing a batch of nanostructures is also inexpensive, Wang said, and since the star structure is so sensitive, the diagnostic test only needs about 5 microliters. The upper bound of the cost per test is $.15, Wang said, compared to the typical $60 cost of RT-qPCR.
The signal can be read by a portable fluorometer, which "opens the door to use the technology in field-based or low resource-based settings," without sophisticated equipment or professional expertise. The target market for the testing is therefore the point-of-care space, Wang said.
He added that dengue is considered one of the most challenging viruses in terms of the structure and arrangement of surface epitopes, but others, like influenza are much simpler. The team has some data on detecting flu, which happens to have inter-cluster pattern on its surface that can help increase the binding specificity as well and reduce crosstalk with other viruses, he said.
The team can also use other ligands, like oligosaccharides, small molecules, or peptides, attached to its DNA structures, to control specificity.
For example, "Different cancer cells have different proteins on the surface, and small molecules are already evolved to sense these epitopes," Wang said.
Bloodstream infections are another potential application, with the high sensitivity of the method potentially enhanced using sample concentration technologies. However, Wang said that rather than go after the pathogen, a better method might be to use the pattern-matching of the DNA sensor method to recognize host-response biomarkers, and the team has begun looking into this application, as well.
The method also has the potential to be used as a therapeutic, since the strong binding of the DNA nanostructure essentially physically isolates the virus. In the Nat Chem study, the researchers showed that being bound to the DNA star inhibited dengue virus from entering cultured liver cells.
The team has further investigated this therepeutic strategy — called pattern recognition-enabled sensing and treatment, or PEST — in cell culture, but how the DNA nanostructures and ligands would behave in the body still needs to be determined. The researchers are now looking for partners to test it on animal models. For some therapeutics, DNA origami might be preferable, Wang said, since the structure is more stable, but the downside is that method is more expensive.
For the dengue test, the surface cluster detection cannot distinguish between the four subtypes of the virus, so the market may be limited to screening or early testing, Wang said, with other tests, like PCR or ELISA, still needed for epidemiology.
The team has filed a provisional patent on the method and is pursuing avenues to commercialization. Wang said he is now looking for a business partner to commercialize the method himself, but if commercial entities want to use the strategy it might also be licensed.