Agriculture Reference
In-Depth Information
than the level in the sprouts and low levels of pathogens may be missed (Fu and others
2001). However, if the testing of spent irrigation water is conducted at 48 h after the
commencement of sprouting, as recommended by the FDA, the level of pathogens
present in the irrigation water will be at a maximum level (Fu and others 2001;
Splittstoesser and others 1983; Stewart 2001a,b) and may be more readily
detectable.
For testing irrigation water, the FDA (1999) has recommended using VIP EHEC
(Biocontrol Systems, Bellview, WA) or Reveal
E. coli
0157:H7 tests (Neogen Corp.,
Lansing, MI) for the detection of
E. coli
0157:H7, and Assurance Gold
Salmonella
EIA or Visual Immunoprecipitate (VIP) assay for
Salmonella
(both from Biocontrol
Systems, Inc., Bellview, WA) for the detection of
Salmonella
spp. However, the
E.
coli
tests require an overnight incubation in modifi ed buffered peptone water with
three added antibiotics, and the
Salmonella
methodology requires preenrichment and
enrichment for approximately 48-50 h before testing. Thus, there is a need to develop
sensitive and specifi c alternatives that can be completed in shorter time periods.
Time-Resolved Fluorescence of Lanthanide Cations
The involvement of 4f orbitals in the electronic structure of lanthanide (La) cations
such as europium permits a transfer of excitation energy from ligands to central La
cations prior to the emission of ion fl uorescence that is characteristic by a relatively
long fl uorescence half - life (
200 nm)
between the absorption and emission maxima. In contrast, the fl uorescence half - life
and Stoke's shift of common organic and biochemical compounds are in the range of
1 - 1000
∼
50 - 1000 msec) and a considerable Stoke ' s shift (
>
sec and 20-100 nm, respectively. Thus, with a pulsed excitation, the fl uores-
cence of La may be easily separated from the interference fl uorescence and scattered
excitation light by delaying the emission measurement (time-resolved fl uorescence,
TRF). In addition, the quantum yield of La-chelates is usually quite high, e.g., 0.18
for Eu - (4,4,4 - trifl uoro - [Z - thienyl - l,3 - butanedionato]) at 614 nm (Halverson and others
1964 ). A combination of time - delayed fl uorescence and the unique properties of La-
chelates has led to the development of a new technique called
dissociation - enhanced
lanthanide fl uoroimmunoassay (DELFIA)
. In this technique, antibodies are modifi ed
to contain binding groups capable of forming very low fl uorescence La - complex. The
modifi ed antibodies are used to capture target species and the antibody-bound La
cations are then extracted out by an “enhancement solution” that contains chelates
capable of forming strongly fl uorescent products (Tu and others 2001b).
μ
Time-Resolved Fluorescence Approach (TRF)
We have applied TRF measurement in combination with immunomagnetic capture to
develop a sensitive and rapid method for pathogen detection (IMB-TRF). This
approach has been demonstrated to detect
E. coli
and
Salmonella
in ground meats at
a 1 CFU/g level and to show that nontarget microorganisms do not interfere with the
detection (Tu and others 2002). In the experiment, europium (Eu) or samarium (Sm)
labeled antibodies to the bacteria were incubated with IMB captured target organisms.
Excess labeled antibody was washed away and the remaining beads were eluted into
an “enhancement buffer,” which contained chelators that extracted Eu or Sm to form
strongly fl uorescent products. The samples were then read in a Victor 1420 Multilabel
