724 Part VII: Food Safety
about 500 cfu/mL in pure culture, and 10^3 –10^4
cfu/25 g of artificially inoculated skim milk (Koo
and Jaykus 2003). The total time of end point detec-
tion is about 2.5 hours (Koo and Jaykus 2003).
Another way to eliminate the need for agarose gel
electrophoresis is use of real-time PCR in a 96-well
PCR format. In this method a fluorescent dye, such
as SYBR Green I, is used to follow the PCR amplifi-
cation in real time and can be used to detect the
amplified products from a number of genes at the
same time (Bhagwat 2003). The genes/pathogens
present can be assigned from the melting curves of
the PCR products. This protocol also allows multi-
ple pathogens to be detected from a universal en-
richment media. For instance, Bhagwat (2003) was
able to simultaneously detect L. monocytogenes, S.
typhimurium, and E. coliO157:H7 from produce.
Including time for culture enrichment, the entire
process took around 30 hours, and allowed detection
limits of 1 cell/mL for E. coliO157:H7 and Sal-
monellaspp. and 10^2 –10^3 cells/mL for L. monocyto-
genes. Hough et al. (2002) were able to detect
between 10^3 and 10^10 cfu for Listeriain 25 g sam-
ples of cabbage without any culture enrichment. Be-
cause of the lack of enrichment, the entire process
from extraction to real-time PCR results was around
8 hours (Hough et al. 2002).
Multiplex PCR is a variation of the traditional
PCR. This method makes use of multiple sets of
primers to amplify a number of genes or gene frag-
ments simultaneously. For instance, Bhagwat (2003)
artificially contaminated produce, cultured the patho-
gens, then used multiplex PCR to simultaneously de-
tectL. monocytogenes,S. typhimurium, andE. coli
O157:H7. Fratamico and Strobaugh (1998) also used
the method to detectSalmonellaandE. coliO157:H7
in bovine feces, carcass wash water, apple cider, and
ground beef that had been inoculated with the two
organisms. After culture enrichment, the authors
were able to detect the pathogens at a level of 1 cfu/
mL (or g), with a total processing time of 30 hours
(Fratamico and Strobaugh 1998). This identification
procedure is effective, but not conducive to high-
throughput screening because of the need to analyze
the PCR products by agarose gel electrophoresis.
In a more elaborate variation for multiplex PCR,
Dunbar et al. (2003) amplified portions of the pro-
karyotic 23S rRNA using universal primers labeled
with biotin. After amplification, the PCR product
was mixed with fluorescently labeled microspheres
displaying pathogen-specific oligonucelotides (see
Fig. 31.3). Each type of microsphere has a different
spectral pattern, and this allowed each type to dis-
play a DNA probe specific for a different DNA se-
quence derived from a different pathogen (derived
from the PCR step). The pathogenic DNA is detect-
ed by adding microspheres specific for each path-
ogen’s DNA. The bound microspheres are labeled
with another fluorescent label by adding streptavidin-
R-phycoerythrin, which binds to the biotin label on
the amplified DNA and fluoresces at a different
wavelength than the microspheres. Because the
spectral label pattern of each type of microsphere is
different, each can be sorted according to its spectral
signature (Dunbar et al. 2003). The amount of fluo-
rescence from the phycoerythrin can be measured
after sorting to determine the quantity of pathogen
DNA present in each species. Theoretically, this sys-
tem could be used to simultaneously detect hun-
dreds of different pathogens. Using the Luminex
LabMAP (Luminex, Austin, Texas) system, the au-
thors could detect L. monocytogenes, E. coli, Sal-
monella,and C. jejunisimultaneously at levels of
103 to 10^5 organisms (Dunbar et al. 2003). The
advantage to this system is that after enrichment and
DNA amplification, enumeration of the original num-
ber of pathogens takes only 30–40 minutes (Dunbar
et al. 2003), and multiple pathogens are detected
simultaneously. However, this system is prohibitively
expensive, still requires time for culturing, and may
experience difficulties with matrix effects.
To address the need of detecting only living path-
ogens, one can amplify the pathogen RNA rather
than DNA. The presence of specific RNA sequences
is an indication of live cells. This process works be-
cause RNA is much less stable and tends to degrade
fairly quickly outside living cells as compared with
DNA. Thus, when an organism dies, its RNA is
quickly eliminated, whereas the DNA can last for
years, depending on storage conditions. Reverse
transcription-PCR (RT-PCR) makes use of a reverse
transcriptase that is able, in the presence of a com-
plementary primer, to create complementary DNA
(cDNA) from an RNA strand corresponding to a
transcribed gene. The cDNA is then amplified using
PCR primers and DNA polymerase under normal
PCR conditions. Analysis of the results is done in
the same way as for PCR.
Klein and Juneja (Klein and Juneja 1997) used
RT-PCR to detect L. monocytogenesin pure culture