Food Biochemistry and Food Processing

58 Part I: Principles

organs, such as the kidney (Riley et al. 1983). In
North America, the O157:H7 strain is found mostly
in the intestines of healthy cattle. The Center for
Disease Control (CDC) estimated that Shiga toxin–
producing E. coli(STEC), such as O157:H7, causes
73,480 illness and 61 deaths per year in the United
States alone and that 85% of these cases are attrib-
uted to foodborne transmission, especially from
ground beef, unpasteurized milk, and roast beef
(Mead et al. 1999). As reports of E. coliO157:H7
outbreaks have become more common, greater
efforts have been made to develop fast and reliable
methods for its detection. PCR has become the
method of choice for pathogen detection because,
contrary to culture isolation and serological tests,
PCR methods provide fast, accurate, and highly sen-
sitive results. Among the genes currently used as tar-
gets for PCR amplification of O157:H7 are Shiga
toxin (stx)(Brian et al. 1992), intimin (Gannon et al.
1993), enterohemorrhagic E. colihemolysin (Hall
and Xu 1998), and -glucuronidase (EC 3.2.1.31)
(Feng 1993). In traditional PCR methods for detec-
tion of pathogens, which include gel electrophore-
sis, the number of samples that can be analyzed dur-
ing one electrophoresis run is very small. For this

reason, researchers from the Department of Nu-

trition and Food Science, University of Maryland,

College Park, Maryland, and from the Center for

Food Safety and Applied Nutrition, Food and Drug

Administration, Washington, D.C., developed a sim-

ple, rapid, large-scale method for the analysis of

PCR products with the use of enzyme-linked

immunosorbent assay (ELISA) (Ge et al. 2002).

This PCR-ELISA approach for the detection of E.

coliO157:H7 and other STEC in food was based on

the incorporation of digoxigenin-labeled deoxyuri-

dine triphosphate (dUTP) and a biotin-labeled

primer specific for stx1and stx2genes during PCR

amplification. In this method, the biotin-labeled

PCR products were bound to microtiter plate wells

coated with streptavidin and then detected by

ELISA using an anti-DIG-peroxidase conjugate

(Fig. 3.15). To establish the specificity of the

primers used in this PCR method, 39 different bacte-

rial strains, including STEC and non-STEC strains

such as Salmonella, were used. All of the STEC

strains were positive and all non-STEC organisms

were negative. The researchers observed that in

comparison with the traditional gel electrophoresis

with ethidium bromide staining method, the ELISA

Table 3.2.Artificial Triplet Codons Encoding for Specific Alphabetical and Numeric Characters,
Used in the Identification of GM Organisms

Characters Codons Characters Codons

1 TTA CCT H CAC AGA

2 TTG CCC I CAA AGG

3 CTT ACC J GTC

4 CTC ACA K GTA

5 CTA ACG L CAG AGT

6 CTG ACT M GTG

7 ATT GCA N AAT GGA

8 ATC GCG O AAC GGG

9 ATA GCT P TCT

0 TTC CCG GAG Q TCC

space TTT CCA AAG R AAA GGT

S GAT TGC

A TAT CGA T GAC TGA

B AT G U GAA TGG

C TAC CGG V TCA

D TAA CGT W TCG

E TAG CGC X GCC

F GTT Y TGT

G CAT AGC Z GGC

Source:Adapted from Marillonnet et al. 2003.