A fluorometric immunological method with a magnetic concentration step was developed for rapid bacteria detection with high sensitivity and specificity in less than two hours without enumeration.
The method involves performing an in-situ immunoassay on a magnetic bead through the formation of a sandwich complex of the target bacteria and the probe followed by the release of fluorophores by enzymatic digestion with proteinase K.
Factors critical to the suitability of detection methods and sensors were investigated, including sensitivity, limit of detection (LOD), detection time and ease of use.
It consists of an immunomagnetic bead as a solid support for capturing bacteria and a probe antibody-dBSA conjugate labeled with fluorophores (IFdBSA).
Pathogens tested
The LOD was <5 CFU/mL of the tested pathogens (E.coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes) in buffer.
When the pathogens were inoculated in foods (spinach, chicken, and milk), the LOD was under 5 CFU/mL for the pathogens.
The procedure of the immunoassays can be performed on a magnetic bead coated with capture antibody, which is specific to the target bacteria.
Upon incubation and binding of the beads to the pathogens, the bead complex is isolated by an magnetic separation step and the target pathogen detected by binding with the IFdBSA conjugate followed by enzymatic digestion for fluorometric signal measurement.
“This in-situ fluorescent immuno-bead assay could be performed in one tube which enables us to not only detect the target bacteria with simple methods but also in very low numbers due to fluorescence enhancement from the large number of fluorescent signal tracers labeled with dBSA used as a carrier of fluorophores,” said the researchers.
Lisa Mauer and Joseph Irudayaraj, two authors of the paper, conducted earlier research to reduce time taken to detect E.coli O157:H7 in ground beef.
Conventional methods
Conventional methods include: colony culture, polymerase chain reaction (PCR), and immunoassays (eg., enzyme-linked immunosorbent assays (ELISA) and immunochromatographic assays), in combination with selective enrichment.
However, these methods are time-consuming, laborious, and/or lack in sensitivity due to the requirement of cultivation steps, said the researchers.
To test the methods for pathogen detection in whole foods, the bead assay was applied to the three different food matrices.
Spinach wash for E. coli O157:H7, boneless chicken for S. typhimurium, and 2% reduced fat milk for L. monocytogenes using uninoculated foods as controls and artificially inoculating each bacteria into the corresponding sample medium.
Bacteria concentrations used, as enumerated by colony counting were: 0.3 and 22 CFU/mL for E. coli O157:H7, 0.5 and 39 CFU/mL for S. typhimurium and 0.4 and 30 CFU/mL for L. monocytogenes.
A 10g sample of chicken was mixed with 10 mL of sterilized phosphate buffered saline (PBS) and inoculated with 1 mL of prepared S. typhimurium; 10 mL of spinach wash was inoculated with 1 mL of E. coli O157:H7; and 10 mL of milk was inoculated with 1 mL of L. monocytogenes.
After inoculation and two hours of incubation at room temperature, 1 mL of the bacterial solution was used and the in-situ bead assays were performed in triplicate.
An extremely low concentration of E. coli O157:H7 (<5 CFU/mL) in spinach wash could be detected by the bead assay and a very high LOD was shown when it was applied to S. typhimurium (>5 CFU/mL) and L. monocytogenes (<5 CFU/mL).
The researchers said it is evident that the analytical sensitivity of the fluorescent immuno-bead assay is high, over 104-fold increase in sensitivity, than conventional colorimetric immunoassays such as ELISA (105–106 cells/mL LOD is common).
Such high level of sensitivity was possible because of the high fluorescent signals obtained from the IFdBSA conjugate, they added.
Source: Biosensors and Bioelectronics, Volume 57, Pages 143-148
Online ahead of print, DOI: 10.1016/j.bios.2014.02.012
“In-situ fluorescent immunomagnetic multiplex detection of foodborne pathogens in very low numbers”
Authors: Il-Hoon Cho, Lisa Mauer, Joseph Irudayaraj