NEW YORK (GenomeWeb) – A team comprising researchers at Ben-Gurion University and Massachusetts Institute of Technology has developed a new method for PCR-based detection of pathogens in soil, water, and aerosol samples that is faster than standard techniques.
The method relies on an initial enrichment of samples and a technique called most probable number, or MPN.
In a study published last month in the journal Water, Air, and Soil Pollution, the MPN-qPCR method was able to detect pathogen concentrations as small as one colony forming unit per gram in environmental soil samples. Detection could be accomplished within a day, compared to about five days for typical pathogen culturing.
MPN is a well established microbiological technique the involves distributing a sample into growth medium such that the concentration of microorganisms can be statistically derived, Ezra Orlofsky, first author on the study told GenomeWeb in an email.
The lab had been using the MPN method to detect "indicator bacteria," such as fecal coliforms and Enterococci in other work, but it was simultaneously "beset by the difficult task of preparing environmental samples for use with qPCR," Orlofsky said.
"It was natural for us to try and integrate the robust statistical approach of MPN with the sensitivity and specificity of qPCR," he explained.
In the method, a pre-enrichment incubation was followed by serial dilution of the broth, and the MPN method was used such that the pattern of present and absent results in each well obtained by subsequent qPCR could then be converted to quantitative data using MPN tables or calculators.
Bacteria can be fussy about their broth, and specific strains can sometimes require their own protocols. However, "We were elated to find that the same conditions could be used to detect and quantify numerous pathogens with almost no additional work," Orlofsky said.
The group used the method initially to detect spiked-in Salmonella enterica and Pseudomonas aeruginosa in sandy and clay soils, wastewater, and a model system system of agricultural produce using the surfaces of tomatoes. They also measured lab-made aerosols.
They were able to show a limit of detection in soil of one to three colony-forming units per gram, five CFUs per tomato, five per liter of treated wastewater, and 300 per milliliter in aerosols.
Once the method was perfected in lab-made samples, the researchers used environmental samples.
The qPCR method employed TaqMan probes and amplification on the Bio-Rad CFX-1000 thermal cycler.
Pre-enrichment using culture methods is being used by researchers in Sweden to enable phenotypic testing in PCR-based assays, as recently reported by GenomeWeb.
Comparing the research, Orlofsky said that "the basic idea is the same; general boosting of bacterial population followed by molecular, as opposed to biochemical detection." He further noted that the growth medium in MPN-qPCR method can be saved for further testing and characterization, such as for antibiotic resistance.
Like the Swedish method, the Ben-Gurion University group also used very simple DNA extraction, in this case by boiling in hydroxide in the presence of SDS.
However, the Swedish group was using the technique to initially measure bacteria in urine and used magnetic beads in the process.
"This is clearly not an economically viable screening approach in environmental samples, where native magnetic debris would overwhelm the system," Orlofsky said.
For infectious disease detection, another recently reported method used lyophilization to concentrate pathogens from nasopharyngeal swabs, but this could also potentially concentrate PCR inhibitors as well.
The BGU researchers get around inhibition by "the synergistic combination of the statistical power of MPN, dilution, and simultaneous growth of the microbes," Orlofsky noted.
The group is now further optimizing the method to reduce incubation periods and expand detection options.
The lab is also working on many environmental projects, such as the application of treated sewage effluents on crops in Israel and Gaza, and Orlofsky highlighted that the method is useful for giving a holistic perspective on microbial contamination of the soil and crops.
"I hope that the method will be of benefit first and foremost to the researchers struggling with the same problems we faced, and the research was published with this goal in mind," he said.
However, he also noted that it is possible the best way to make the process accessible may be to commercialize it, and co-author Osnat Gillor, a researcher in the Department of Environmental Hydrology and Microbiology at BGU, would be open to inquiries.
Overall, the integration of fast-culture and PCR is much more objective and definitive than biochemical identification, and Orlofsky predicted that a few simple culturing conditions could enable testing of most known bacterial pathogens. Coupling isothermal PCR and dyes that do not require fluorescence could reduce the need for electricity, he noted. Methods using digoxigenin or biotin, or dyes recently developed by New England Biolabs that use protons released from the PCR reaction for pH-sensitive readouts, might be useful, for example.
"Housekeeping genes for all known bacterial pathogens are well known and characterized, so we are really on the cusp of low-cost, rapid, and universal pathogen detection in environmental samples," Orlofsky said.
"The extent and utility of this approach is yet to be fully realized," he said.