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Paper DNA Detection Device Enables Diagnosis of Helminth Infections

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NEW YORK (GenomeWeb) – Researchers at Brock University in Canada have developed a low-cost assay to detect soil-transmitted helminth infections. The test uses a quantitation technique involving capillary flow that exploits an interaction between the SYBR Green I dye and cellulose filter paper the researchers claim has never been described before.

A description of the quantitative paper-based DNA reader (qPDR) and its use on clinical samples from pediatric cases of helminth infection in Honduras was published last month in ACS Sensors.

Feng Li, a bioanalytical chemist at Brock and co-author of the study, said that the interaction between SYBR Green I and cellulose paper was a "surprising" phenomenon. Under the right conditions, Li said, SYBR Green I binds tightly to cellulose paper and emits fluorescence upon binding. "Those are two phenomena that have never been reported before," he said. 

SYBR Green I also tightly binds double-stranded DNA (dsDNA), but not single-stranded DNA, and binding strength between the dye and dsDNA is stronger than that between the dye and cellulose paper, Li explained.   

Thus, after loading sample dsDNA onto filter paper saturated with a high concentration of SYBR Green I, the DNA will migrate because of capillary forces and the dye will be ripped off the cellulose and carried along because of a stronger binding between the dye and dsDNA. 

And, according to the study, because the eluent strength of dsDNA is concentration dependent, it is possible to quantify the amount of dsDNA in a sample by simply reading the migration distance of SYBR Green I, given prior calibration with standards of known concentration.

Li noted that the original motivation leading to these insights came from a collaboration with Ana Sanchez, a health sciences researcher at Brock University specializing in neglected parasitic diseases who studies soil-transmitted helminth infections in Honduras. 

Infections with parasitic soil-transmitted helminths affect about 1.5 billion people worldwide and are a major cause of childhood malnutrition and physical impairment, according to the World Health Organization

Although WHO recommends periodic large-scale deworming with inexpensive and easy-to-administer medications to control infections in endemic areas, an inexpensive diagnostic could also be useful, Li said. Helminth infection is not a very deadly disease, he said, but the chronic malnutrition it can cause is dangerous. A mild infection might be managed without treatment for a while, but a massive infection requires immediate treatment.

Unfortunately, lack of lab facilities in endemic areas of Honduras is a big issue, and to get qPCR results, healthcare workers must ship samples to labs as far away as the US and Canada. This can lead to weeks-long turnaround times after field testing.

Brainstorming ways to do a quantitative DNA test without a qPCR instrument led Li and his colleagues to the paper-based strategies, and failed attempts to make paper chambers to contain SYBR Green I resulted in the discovery of the unique interaction between the dye and cellulose paper.

"Based on our understanding of this chemistry, we modified the assay and eventually, it became a distance-based quantitative tool," Li said.

The test requires an amplification step prior to using the qPDR reader, but it uses a portable thermal cycler for this. Specifically, the study used the MiniPCR system from Amplyus, a small instrument that has recently been used to amplify DNA aboard the International Space Station.  

However, the cellulose reader is compatible with any PCR instrument, as well as with loop-mediated amplification or other isothermal technologies, Li said. Reading the results of the test with the qPDR would only require a simple UV lamp or a blue light box. And, the method could be extrapolated to detect DNA from other parasites or pathogens as well.  

Lateral flow strips are a competing technology, Li said, but qPDR has two advantages. Typical lateral flow strips require primers to be modified so they can capture dsDNA, Li said. "In our case, we don't rely on any specific capture, so we don't need to modify the primers, which could potentially reduce the cost per assay." The other advantage over lateral flow strip is the quantitation, since lateral flow tests can only provide "yes or no" results, he added.

The researchers have applied for intellectual property protection and are looking at different opportunities for commercialization, for example through a spinout company or an industry collaboration. "We want the technology to be used by patients," Li said.