Agriculture Reference
In-Depth Information
Because
E. coli
0157:H7 bacteria can be a hazard on cantaloupe from the fi eld as
well as in postharvest handling, rapid and sensitive detection methods are needed. To
determine the levels of
E. coli
0157:H7 on cantaloupe, we tested the applicability of
two different detection methods (Fig. 18.1) involving the use of immunomagnetic
heads to fi rst capture and concentrate
E. coli
0157:H7 from cantaloupe samples. The
captured bacteria (shown as B in the fi gure) were than detected either by the biolu-
minescence of cellular NAD(P)H or by a chemiluminescent sandwich assay. The
results showed that the methods developed were capable of detecting relatively low
levels of the
E. coli
0157:H7 spiked on the surfaces of cantaloupes within 3.3 h (Tu
and others 2004 ).
NAD(P)H Method
The NAD(P)H method mentioned above involves the measurement of cellular NAD(P)
H via an externally added electron transfer system that uses membrane permeable
menadione to oxidize internal NAD(P)H to NAD(P)
+
. Menadione reduces molecular
oxygen to hydrogen peroxide (H
2
O
2
) that generates chemiluminescent luminol by the
action of horseradish peroxidase (Fig. 18.2). This reaction system utilized the cellular
NAD(P)H and membrane-bound electron transfer process to produce luminol-
supported and peroxidase - catalyzed chemiluminescence.
As described in our previous report (Tu and others 2004), the NAD(P)H method
was useful to measure the presence of viable cells. Figure 18.2 shows the method
NAD (P)H Method
Sandwich Method
IMB
IMB
B
B
NAD (P)H in cell
Catalyses reaction
Peroxidase labeled
antibody
menadione
menadiol
H
2
O
2
+
Luminols
+
O
2
-
Luminols
O
2
Luminescence, h
n
+
oxidized products
Peroxidase
Figure 18.1.
Two luminescent assays for bacterial detection. The bacteria (B) either
provide the cellular reducing power in the forms of NAD(P)H or are sandwiched with
peroxidase labeled antibody.
