Web Date: March 29, 2011
Speedy Microarray For Harmful Bacteria
To prevent outbreaks of water- or food-borne illnesses, scientists need rapid, sensitive methods to detect pathogenic bacteria such as Escherichia coli, Salmonella enterica, and Campylobacter jejuni. Now researchers report a microarray assay that uses chemiluminescence to spot these three bad bugs (Anal. Chem., DOI: 10.1021/ac2002214).
Currently, scientists look for harmful bacteria in drinking water samples by growing cultures of microbes, by using antibody microarrays to snag specific species, or by using a real-time polymerase chain reaction. But culture methods are time consuming, antibodies have trouble detecting bacteria below certain concentrations because of their relatively low sensitivities for molecules on bacteria's surfaces, and real-time PCR can check for at most dozens of species.
So Michael Seidel and his colleagues at the Technical University of Munich developed a new system that identifies pathogens by their DNA sequences. Their method starts with lysing cells from a sample and amplifying the cells' DNA by running a shortened PCR procedure. They tagged the resultant DNA with digoxigenin, a steroid that antibodies can recognize with high sensitivity.
The scientists then squirt the digoxigenin-tagged DNA onto a microarray containing characteristic DNA sequences from the three pathogenic bacteria. After the researchers allow time for complimentary DNA strands to bind, a step called hybridization, they add an enzyme-labeled digoxigenin antibody to the microarray. This enzyme produces a chemiluminescent signal when the researchers add the dye luminol and peroxide. If the sample contained any pathogenic bacteria, the microarray spots corresponding to that species' DNA would then light up.
Seidel and his colleagues found that their method is as sensitive as the best real-time PCR methods: It could detect Campylobacter jejuni at concentrations down to one cell per mL of water, and Salmonella at 500 cells per mL of water.
Although the workflow has a lot of steps, it takes under four hours from start to finish. Chemist Sylvia Daunert of the University of Kentucky, calls that speed "a significant improvement over other detection methods." Commercial methods can take up to 24 hours.
The method's speed comes from its use of microfluidics to speed up hybridization, the slowest step. Instead of dripping the PCR products onto the microarray and just letting it sit there, the German team set up an injection system that sloshes it back and forth repeatedly to reduce the hybridization time.
Another key is the shortened PCR reaction. Instead of running the typical 40 to 50 cycles of the reaction, the German team ran 30 to 35. More cycles produce more DNA strands, which leads to increased sensitivity, but note.
In comparison to antibody microarrays, the technique is quite sensitive, Daunert says. But it can give inconsistent concentration readings at low levels of bacteria, she says. For these dilute samples, it may be necessary to use filters or magnetic beads to concentrate the germs.
Trevor Suslow, a produce safety expert at the University of California, Davis, says that a rapid detector for multiple pathogens is "both badly needed and of great interest." But he cautions that several microfluidic systems have worked well in pure water and then failed during real-world evaluations.
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