Agriculture Reference
In-Depth Information
detect small amounts of target nucleic acid in a sample (Toze 1999). PCR has been
shown to accurately detect low numbers of microbes such as viruses (Schwab and
others 1996) and bacteria (Jensen and others 1993; Fach and Popoff 1997). Multiple
primers can be used to detect different pathogens from one multiplex reaction.
However, this technique can be limited by problems such as the sensitivity of the
polymerase enzyme to environmental contaminants, diffi culties in quantifi cation, gen-
eration of false positives through the detection of naked nucleic acids, nonviable
microorganisms, or contamination of samples in the laboratory (Toze 1999). Nucleic
acid-based assays can indicate only the genetic potential of a microorganism to
produce toxin or to express virulence and do not provide any information on toxins
in foods or environmental samples. From a practical point of view, the routine detec-
tion of microbes using PCR can be expensive and complicated, requiring highly
skilled workers to carry out the tests.
All of these methods require samples of small volume and they may have the
sensitivity to detect infectious dosage of pathogens in the sample (e.g., only a few
cells of
E. coli
O157:H7). However, the low levels of pathogen may not be uniformly
distributed, but highly localized in a food matrix. Assuring that the samples used for
detection contain the target pathogen is a demanding challenge. To circumvent this
problem, culture enrichment of multiple samples is often needed to increase cell con-
centration and thus enhance pathogen detection.
Alternatively, immunomagnetic beads (IMB) can be used to rapidly and effectively
separate and concentrate targeted pathogens, and the method has attracted increased
interest (Molday and others 1977; Sinclair 1998). IMB have been applied in several
rapid methods to capture pathogens prior to analysis (Fratamico and others 1992;
Olsvik and others 1994). IMB also have been used to increase the signal intensity by
concentrating captured pathogens into smaller detection volumes (Gehring and others
1996; Yu and Bruno 1996). In the past, we developed detection processes that involved
fi rst capturing targeted pathogens in foods from briefl y enriched cultures by the use
of specifi c IMB (Tu and others 2001a). The captured pathogens were further conju-
gated with second antibodies labeled with signal generating tags. The sandwiched
complexes involving IMB, targeted pathogens, and labeled antibodies could be pro-
cessed by the use of suitable magnetic devices. The captured pathogens were then
revealed by different optical and electrical approaches. With this general approach,
we were able to detect
1 CFU/g of target pathogens in meat samples in a standard
8-h shift (Tu and others 2001b). Some of those developed approaches were also
applied to produce systems. In the following sections, we briefl y summarize our
experience of detecting pathogens in produce by IMB and biosensors.
∼
Biosensor Processes Involving the Use of IMB for
Pathogen Detection
For these approaches, micron-sized iron-containing beads coated with antibodies
specifi c to antigens of target organisms form IMB that are used to capture those targets.
The captured pathogens and excess IMB can be easily separated from other solution
components and conveniently transferred to a desired detection environment by the
use of high-strength neodymium boron iron magnets associated with an automated
