The presentation by Sam Nugen, a graduate student in Cornell’s food science department, focussed on detecting the food-borne bacteria Streptococcus pyogenes (S. pyogenes), but the technique can be applied to a wide variety of bacterial pathogens, including Escherichia coli (E. coli).
The new biosensor works in a test tube and a positive result shows up as a red line on a strip, much like a pregnancy test. Newly designed software gives researchers a powerful tool for increasing the sensitivity of the analysis.
The method may help researchers and companies that are in the business of tracking food-borne pathogens, allowing technicians to determine a source quickly. It may also help to analyse a throat culture swab, to tell if someone has an illness like strep throat.
“We hope to see this technique commercialised, because it is very rapid compared to all the standard methods right now,” said Nugen, the study’s lead author. Nugen conducted his research in the laboratory of Antje Baeumner, Cornell associate professor of biological and environmental engineering, who is also a co-author of the study. “It would be great if we came up with something that became a standard,” Nugen added.
Current biosensors rely on a time-consuming technique called gene amplification that requires costly equipment: Technicians take a piece of DNA from a sample and add enzymes that make many copies of the DNA. Duplicating or “amplifying” the DNA makes a pathogen easier to detect.
The new process starts with genetic material that is extracted from a food sample. This material, called ribosomal RNA (rRNA), is responsible for translating genetic information carried in DNA into proteins.
Nugen designed the computer software that allows researchers to enter in an rRNA sequence, called a target sequence, that is unique to a specific microbe. The program then determines tiny sequences of complementary DNA, known as probes, that are exactly matched to stick to the rRNA target sites. These sequences are then reproduced as genetic material by a biotech company.
To test for the presence of scarlet fever-causing bacteria, a sample of rRNA is placed in a test tube with two of the manufactured probe DNA sequences, designed specifically to bind to the rRNA of S. pyogenes. One of these is called a “capture” probe, and the other is known as a “reporter” probe. The capture probe binds to the S. pyogenes rRNA and anchors it to a zone on a strip of membrane, while the reporter probe, which has a dye attached to it, sticks to another sequence of the S. pyogenes rRNA.
The probes attach to the target rRNA sequence when the test tube is placed in a water bath for about 25 minutes at exactly 41 degrees Celsius (106 Fahrenheit). At that temperature, the complementary probe DNA sequence binds to the target RNA sequences.
In a positive result, the capture probe attached to the rRNA target molecule anchors itself to a strip on a membrane. Since the reporter probe with a red dye is also attached to the rRNA molecule, as the material collects on a zone on the membrane, it turns the strip visibly red, much like with a pregnancy test.
The entire process takes only 35 minutes, while traditional gene amplification techniques may take many hours. Promising early results suggest the sensitive method could detect fewer than 100 cells of a pathogen in just half an hour.
Nugen’s software also compares a target site with sequences from other organisms to make sure they don’t overlap, which could lead to a misdiagnosis.
“You want to be sure it doesn’t detect another organism that shares similar properties,” said Nugen.