Verma, on the other hand, is using qLAMP — quantitative loop-mediated isothermal amplification. It also amplifies a small amount of DNA or RNA in a sample, but it requires a constant 65 degrees Celsius (149 degrees Fahrenheit) and yields results within an hour. All of the primers and chemical reagents needed to run the assays can be built onto special test paper, much like a pregnancy test.
“This makes the device easier to run and easier to build,” he said.
For the project’s first objective, the researchers wanted to establish background levels of animal-borne bacteria near an animal feeding operation. As indicators of fecal contamination, they chose to screen for Bacteriodetes, a group of anaerobic bacteria found in mammal guts. The bacteria already are used as biomarkers of fecal contamination in water.
In addition, the researchers compared sampling methods for detecting airborne contaminants. To do this, they placed pots of greenhouse-grown lettuce or plastic collection flags at different distances from the source. They then swabbed or surface washed the plants to collect samples and ran them using qPCR, since that is the gold standard.
Because of their smooth surfaces, the flags were easier to swab and produced more consistent results compared to the lettuce leaves, Verma said.
The results from the lettuce leaves also were highly variable and had no correlation between the distance from the feeding operation and the amount of contamination.
As part of objective one, the researchers compared the portability of using qPCR and qLAMP. The qLAMP process could be done in the field and provide a result in about an hour, whereas qPCR had to be done in the lab and required several hours.
“What we’ve shown is that Bacteriodetes are good markers, we’ve developed an assay that can change color in the presence of Bacteriodetes, and we’ve shown we can go out and do assays in the field,” Verma said.
The next step, part of the second objective, will be to test 200 samples in the field using microfluidic paper-based analytical devices and a portable detection system. The goal will be to produce field results using qLAMP comparable to the laboratory’s but in under an hour.
Verma said he hoped their work will eventually lead to a commercial portable risk-management tool for growers and packers.
“What we are envisioning in the long term is to build a kit with collection flags to use with these tests — if it gets commercialized,” he said.
