CHAPTER 6

DEVELOPMENT AND IMPACT OF RAPID METHODS FOR DETECTION OF

FOODBORNE PATHOGENS

Introduction

Analysis of foods for the presence of bacteria is a common practice today for ensuring
food quality and safety.

Today, microbial analyses of food samples are routinely performed. However, the
introduction and gradual implementation of the Hazard Analysis and Critical Control
Point (HACCP) concept to safeguard foods “from farm to fork” will probably reduce our
dependence on end product analysis. End product testing is generally regarded as
ineffective in ensuring the safety of foods. But microbiological testing and the demand
for better testing methods will continue, as these assays still play critical roles in food
production, in the establishment and validation of HACCP procedures, and in the
monitoring of food quality to ensure the distribution of safe foods to the consumer.

Currently, there are many microbiological methods to test for most of the pathogens
commonly found in foods. For the most part, these are conventional methods that rely
extensively on culture and agar media to grow and enumerate viable bacterial cells.
However, the advent of biotechnology has greatly altered food testing method, and there
are numerous companies that are actively developing assays that are specific, faster, and
often referred to as “rapid methods,” use technologies such as special fluorogenic or
chromogenic enzyme substrates, monoclonal or antibody cocktails, nucleic acids, and
PCR to develop more efficient diagnostic assays. Some of these are automated.

Problems in Food Analysis

Microbiological analysis of foods, especially for particular pathogen species, remains a

challenging task for virtually all assays and technologies. The problem may be due in part
to the fact that bacteria are not uniformly distributed in foods, but analysis is also
complicated by the heterogeneity of food matrices. Foods are composed of an infinite
array of ingredients that include proteins, carbohydrates, fats, oils, chemicals,
preservatives and many other compounds. The physical form of foods is also highly
variable: foods come in powder liquid, gel, solid, semisolid or other forms. These
differences in viscosity, compounded by the presence of ingredients such as fats and oils
can interfere with proper mixing. This results in nonuniform homogenates which do not
allow reproducible analysis or consistent isolation of specific pathogens.

In addition to the problems of variable matrices, testing of foods is further complicated

by the presence of indigenous microbiota. In some raw foods, such as sprouts,
populations are as high as 108 cells/g. Normal microbiota generally pose no significant
health risks, but their presence often interferes with the selective isolation and
identification of specific pathogens, which are usually found in much lower numbers.
This interference becomes especially critical when foods are being analyzed for
pathogens such Shigella sp. And enterohemorrhagic Escherichia coli (EHEC) of serotype
O157:H7. These pathogens have low infectious dosages, making the consumption of low
numbers of cells dangerous.

The problems caused by normal microbiota and matrix interference are further
exacerbated if the specific pathogen of interest is injured. Many processes or treatments
used in food manufacturing, such as heat, cold, drying, freezing, osmotic activity,
chemical additives, preservatives and other factors can sublethally injure bacteria cells.
These injured or stressed cells are sensitive to many of the compounds or antibiotics used
in selective enrichment media; hence, unless injured cells are allowed to resuscitate, they
may easily be outgrown by other bacteria present in the sample.

Although the procedure for conventional methods described above is effective and has
been used routinely for isolating and identifying bacterial pathogens in foods, the method
is labor-intensive and time-consuming. As a result, conventional methods usually take
several days to perform and are inadequate for making quick assessments of the
microbiological safety of foods.

Development and Origin of Rapid Methods

Scientific advances have introduced a number of technologies that are being used to
detect foodborne pathogens and their toxins. These assays are collectively referred to as
rapid methods, a term that has steadily gained in popularity and interest worldwide.

Definitions of Rapid Detection and Identification

To define exactly what constitutes a rapid method is not simple, as “rapid” is subjective
to interpretation. As a result, a rapid method can be an assay that gives instant or real-
time results, but such ideal rapid methods do not yet exist. On the other hand, a rapid
method can also be a simple modification of a procedure that shortens the assay time.
Since conventional methods, generally regarded as “the gold standard,” often take days in
not a week to complete, any modification that reduces this analysis time can technically
be called a rapid method. Between these two extremes of definitions, therefore, virtually
all new methods that have been developed or are being developed can be classified as
rapid method. So the term “rapid methods” encompasses a large group of diverse
technologies and assay formats.

“Identification” and “detection” are two terms often used in conjunction with rapid
methods that also warrant clarification. Although these terms have used synonymously,
identification usually refers to determining the identity of a pure culture isolate, whereas
detection refers to detecting the presence of an organism directly in foods, without the
need to isolate the organism.
RAPID METHOD FORMATS

Rapid methods are usually more sensitive and specific than microbiological methods for
identification or detection of pathogens in foods. The common formats of commercial
rapid methods are (i) miniaturized biochemical identifications kits, (ii) modification of
conventional microbiological methods, and (iii) antibody-based and (iv) nucleic acid-
based detection assays. Within each group, there are also some automated identification
and detection assays. Under the broader definition of “rapid,” all of these are rapid
methods, since they do expedite the identification or detection process. But if the more
stringent definition is applied, the use of the term rapid methods to describe these assays
may be misleading. For instance, in identification tests, the actual assay may be rapid, but
since a pure culture is required, it still takes time to grow and plate that isolate. Likewise
the complexity of food matrices and the other analysis problems discussed above have
precluded the use of most detection methods directly on food samples. As a result, almost
all rapid methods designed to detect a specific pathogen still require enrichment of the
food samples in media prior to testing. In other words, the performance of the assays
themselves may be very rapid, ranging from a few minutes to a few hours, but the total
time for analysis of a food sample requires a few days or longer because of cultural
enrichment. Despite the need for enrichment, however, many rapid methods use
abbreviated enrichment steps and eliminate the need for deferential plating for colony
isolation. So there are still significant reductions in overall assay time as compared with
conventional methods. The technologies and the many unique designs used in rapid
methods are discussed in the following sections.

Miniaturized biochemical and other identification assays

Bacteria isolated from foods are identified by their biochemical characteristics. To

generate a biochemical profile is a labor-intensive, media-consuming process, in which
pure cultures of bacteria are subjected to a battery of tests to determine various enzymatic
activities and the utilization of assorted carbohydrates. The initial concept to miniaturize
biochemical testing began in 1940s, when the use of small vials greatly economized
media usage, and the use of concentrated inocula considerably reduced incubation time.
Since inception, miniaturized biochemical tests steadily gained in popularity in clinical
microbiology, and later, the benefits of using these kits in food analysis became apparent.

Miniaturized biochemical test Kits are used for the identification of pure cultures of
bacteria isolated from food. Most Kits consist of a disposable, multichamber device,
containing 15 to 30 media or substrates, designed to identify a specific group or species.
With the exception of a few systems, in which results can be read in 4 h, most Kits
require 18 to 24 h of incubation. In general, miniaturized biochemical tests show 90 to
99% accuracy and are comparable to conventional identification methods. Although most
Kits are designed fro enteric bacteria, biochemical Kits for the identification of non-
Enterobactriaceae are also available, including Kits for Campylobacter, Listeria,
anaerobes, nonfermenting gram-negative organisms and gram-positive bacteria.
Miniaturized biochemical identification Kits are all very similar in procedure, cost, and
performance and are certainly much simpler and more economical than conventional
methods. However, there may be some differences in the size of the database: the older
its have been used longer and may have a more extensive identification database. The
concept of the minisystems has basically remained the same, but the newer assays are
designed to be more user friendly, with less hands-on manipulation or need for
extraneous reagents, compared with the earlier Kits.

Most automated systems make identifications based on biochemical profiles. In this test,
a disposable, credit card-sized plastic device containing up to 30 biochemical tests is
inoculated with a pure culture suspension of biochemical reactions to generate a
phenotypic profile, which then compared with the computer database to provide
identification.

A few other automated systems identify bacteria based on compositional or metabolic

properties. The microbial Identification System uses gas chromatography to analyze cell
extracts of pure bacterial cultures to generate a fatty acid profile. The computer then
analyzes and calculates the peak data on the chromatograms and compares them with the
database for best-match identification. The Biolog Identification System generates
identification profiles based on the ability of the bacteria to oxidize a panel of 95
different carbon sources. The respiration of viable bacterial cells on these carbon sources
causes changes in the redox potential, which are detected colorimetrically using a
terazolium-based compound. The color changes are measured by spectrophotometer, and
the resulting profile is compared with the reference database in the computer.

Modifications and Specialized Media

Another category of rapid methods includes a large variety of assays, ranging from
specialized media to simple modifications of conventional assays that result in savings in
labor, time and materials. The group includes tests like Petri film, which uses disposable
cardboard containing dehydrated media, designed fro enumerating total bacteria, specific
bacterial species, or mold and yeasts. This test eliminates the need for preparing media
and agar plates, economizes in storage and incubation space, and also simplifies disposal
of materials after analysis. The Isogrid tests uses specially designed hydrophobic grid
membrane filters (HGMF) that can handle larger cells densities. This reduces the number
of dilutions needed prior to filtration. HGMF increases counting capacity from 80
CFU/ml, allowed on normal membranes, to up to 1600 CFU/ml. There are two other
developments that have had a big impact in this area. One was the introduction of
specialized substrates that are used in media or in substrate-impregnated disks to enable
rapid detection of trait enzymatic activities; the other is an assay that enumerates total
bacterial counts based on bacterial ATP.

Color pH indicators have been used in media for years to measure changes in acidity due
to bacterial metabolic activities. However, the introduction of chromogenic and
fluorogenic substrates greatly stimulated the development of special microbiological
media. These substrates provide a quick measure of specific enzyme activities, some of
which are trait characteristics of certain bacteria or bacterial groups, thus providing a
quick presumptive identification of the organism. Some chromogenic substrates, like o-
nitrophenyl-β-D-galactosidase (ONPG) and 5-bromo-4-chloro-3-indolyl- β-D-galactoside
(X-GAL), which was developed for use in molecular biology to detect promoter activities
of DNA fragments cloned into expression vectors, have been incorporated into culture
media to measure β-galactosidase (GAL) activity in enteric bacteria . Similarly, the
fluorogenic substrate 4-methylanbelliferyl- β-D-glucuronide (MUG) has been added to
various media to specifically detect E. coli β-glucuronidase (GUD) activity. Today, many
specialized media, including substrated paper discs, use MUG or other chromogenic
GUD subsubstratesfor detection of E. coli. O

Bioluminescence assays that detect bacterial ATP have also become common in food
analysis. The key components of these assays are the enzyme luciferase, the organic
compound luciferin, and magnesium ions. In the presence of ATP, luciferin is oxidized to
oxylucifein and, in the process, photons are released. By using ATP as the limiting
reagent, the number of photons emitted, which can be quantified with a luminometer,
becomes directly proportional to the estimated number of bacterial cells initially present
in the sample. Luciferase reactions are rapid and, unlike other methods, which require
culture enrichment, ATP assays can detect 104 CFU/ml in 5 to 10 min without the need
for enrichment. These assays are useful for monitoring the sanitary quality of foods or
food processing surfaces; however, since all living organisms produce ATP, they can
only be used to estimate the number of the total bacterial population, not the presence of
specific bacteria or pathogens.

Nucleic Acid-Based Assays

There are many nucleic acid-based assays, but only DNA probe, PCR, and bacteriophage
have been developed commercially for detecting foodborne pathogens. Recently, a
number of DNA-base molecular typing methods, including pulsed field gel
electrophresis, restriction fragment length polymorphism, and ribotyping, have also been
developed. Typing methods are extremely useful for epidemiological investigations and
for characterizing and fingerprinting foodborne pathogens.

DNA Probes

DNA probes for detecting pathogens in foods were introduced in the early 1980s. The
actual commercial growth of probe assays has been unexpectedly slow, perhaps because
of the initial use of radioactive markers in these assays. So, despite the fact that DNA
probes have been developed for most of the major foodborne pathogens, only a few
companies are actively pursuing this format for food testing. Probe assays generally
target rRNA, taking advantage of the fact that the higher copy number of bacterial rRNA
provides a naturally amplified target and afford greater sensitivities.

The GENE-TRAK assay uses a combination of DNA hybridization and enzyme

immunoassay for the detection of foodborne pathogens. The key components are a
polydeoxyadenosine-tailed capture probe and a fluorescein-labeled detector probe. Both
of which are specific for adjacent regions of the target rRNA. After cultural enrichment
of the food sample and chemical cell lysis, the rRNA target is hybridized simultaneously
with both probes, and the hybrid is subsequently immobilized with a polydeoxythymidine
coated dipstick. Unbound or nonspecific binding is removed by multiple washing steps,
and the bound complex is detected by enzyme immunoassay with peroxidase-conjugated
anti-flourescein antibody and a colirimetric peroxidase substrate.

The AccuProbe assay by GEN-PROBE is a hybridization protection assay in which a

single-stranded probe, tagged with a chemiluminescent acridium ester label, is hybridized
to specific sequences in the 16S rRNA. Hybridization protects the probe and label;
unbound probes are inactivated by deferential hydrolysis. Addition of alkaline H 2O2
activates the label to emit light at 430 nm, which can be quantified with a luminometer.
The AccuProbe assay is used mostly for identification of pure culture isolates of bacteria;
however, it has also been explored as a test for pathogens in food enrichment samples.

PCR

Although the use of DNA probes for food analysis has not gained the projected
commercial dominance, the basic principle of hybridization used in probe assays is being
utilized by other technologies. In the PCR assay, short fragments of DNA (probes) or
primers are hybridized to a specific target sequence or template, which is then
enzymatically amplified by Taq polymerase using a thermocycler. PCR is an extremely
powerful tool that enables exponential amplification of a specific sequence in a short
time. PCR assays can be designed in a multiplex format to simultaneously detect several
targets. Theoretically, PCR can amplify a single copy of DNA a million-fold in less than
2 h; hence, it has the potential to eliminate or greatly reduce the dependence on cultural
enrichment.

PCR interference lies mostly with the presence of inhibitors in foods, which can affect
primer binding or amplification efficiency or cause the occurrence of false-negative
results. In the application of PCR for testing foodborne pathogens, foods with high
protein and fat content, such as chicken, meat, soft cheeses and oyster tissue, tend to
interfere most with PCR amplification. This may be due to the presence of proteinases in
these samples, which can degrade the enzyme. In addition to food components, excess
levels of some ingredients used in enrichment or media and compounds used in the
extraction of DNA from food samples can also interfere with PCR. As a result, food
samples or culture-enriched samples often need to be treated or extracted to produce
suitable template DNA for PCR analysis. Furthermore, because of the complexity of
foods, a preparation method developed for a particular food type may not be usable for
other foods. For example, extraction using alkaline conditions and detergent can reduce
PCR inhibitory effects from high fat- and protein-containing foods. But other foods may
require dilutions, washings, filtrations or centrifugation steps to remove inhibitory
effects. Also, immunomagnetic separation, in which antibodies are used to capture and
concentrate specific bacteria, is effective in reducing the inhibitory effects of foods and
has facilitated the use of PCR in testing foods.
Aside of PCR, there are other methods for DNA amplification. Some of these, such as
ligase chain reaction and self-sustained sequence replication (3SR), are being explored
for testing for foodborne pathogens.

Bacteriophage

The highly specific interaction of phage with its bacterial host can be used to detect
specific pathogens in foods. Two examples are bioluminescence and ice nucleation. As
discussed above, assays that measure ATP cannot differentiate bacterial species, so they
are used mainly to quantify total bacterial load in foods or to monitor sanitation. But
bioluminescence assays can be used to identify bacteria if specific bacteriophages are
engineered to carry detectable markers. Several species in the genera Alteromonasi,
Photobacterium, and Vibrio carry the lux operon that encodes bacterial luciferase and
therefore are naturally bioluminescent. The lux genes from Vibrio fischeri were cloned
into Charon 30 phage to develop an assay that is specific for E. coli. Since phages do not
have mechanisms for gene expression, the lux genes are silent. But once the recombinant
phage infects its host, the inserted lux genes are expressed within a short time, resulting
in a bioluminescent host. The amount of luminescence emitted can be quantified with a
luminometer and calibrated to reflect the original bacterial load present in the sample.
The recombinant Charon phage detected as few as 10 E. coli cells in 100 min after
infection and detected 100 to 1000 cells in milk or urine in 1 h, without cultural
enrichment. Similar sensitivities were reported for a lux recombinant phage using pure
cultures of Salmonella sp. However, since the required detection limit for Salmonella is 1
cell in 25 g of food, a short cultural enrichment is still required in bioluminescence
assays.

The Bacterial Ice Nucleation Diagnostic (BIND) assay for Salmonella sp. is another
bacteriophage assay. It uses the ina gene for ice nucleation and is based on the physical
properties of freezing. Small, homogeneous volumes of water can be supercooled to -
40°C and remain liquid; but if nucleating agents are present, ice crystals will form at -
2°C. most ice nuclei are inorganic molecules, but some phytopathogenic bcteria, such as
Erwinia, Pseudomonas, and Xanthomonas spp., produce proteins that also nucleate ice.
Ice nucleation proteins, encoded by the ina gene, seem to bind water molecules to
simulate configuration of ice crystals, thereby promoting freezing. By cloning the ina
gene into a Salmonella-specific phage, the presence of this pathogen in foods can be
detected by using a freeze-indicator dye. The detection limit of the BIND assay is
reported to as low as 20 CFU/ml but ranges between 100 and 10000 CFU/ml, depending
on the Salmonella serovar being detected; hence, enrichment of foods is still needed for
optimal detection. Aside from detection assays, ice nucleation technology is also being
explored by industry to raise freezing times and to improve the quality, flabor, and
texture of frozen foods.

Antibody-Based Assays

Antibody has been used for many years to serotype bacteria. One of the earliest typing
schemes was devised in the 1920s and 1930s to identify Salmonella serotypes.
Development of the radioimmunoassay expanded the use of antibodies to include
quantitative determination of antigens, but it was not until the introduction of highly
sensitive tests, such as the enzyme-linked immunosorbent assay (ELISA), that the
potential of antibodies in detection was realized. The highly specific binding of antibody,
especially monoclonal antibody, and the simplicity and versatility of antigen-antibody
reactions have facilitated the design of a variety of assays and formats. In general,
antibody assays are classed into five formats, and they compose the largest group of rapid
methods used in food testing.

Latex Agglutination and Reverse Passive Latex Agglutination

The simplest antibody test is latex agglutination (LA), whereby antibody-coated colored
latex beads or colloidal gold particles are used to test bacterial cell suspensions. If the
specific antigen is present, visible clumping or a precipitate is formed. Although
agglutination reactions are extremely simple and occur almost instantly, they are not very
sensitive and require about 107 CFU for a reaction. This, however, still represents a 10-to
100-fold increase in sensitivity over the traditional heme or cell agglutination assays. Fro
analysis of bacterial toxins, the assay is known as reverse passive latex agglutination
(RPLA), and it is usually done in a microplate. The main difference is that the antigens
(bacterial cells) in LA are insoluble so that, instead of clumping, the formation of a
diffuse lattice work is considered positive. In food analysis, LA assays have been
evaluated for detecting Salmonella sp.; however, multistep enrichment is needed prior to
testing, and in some cases, suspended food particles affect the interpretation of
agglutination reactions. LA is most useful for quick serological identification or
confirmation of isolates from foods, and RPLA is used mostly for analysis of toxins in
food extracts or for toxin production by pure culture isolates.

Immunodiffusion

Immunodiffusion is another simple antibody assay format used in testing for pathogens in
foods, but the Salmonella 1-2 Test is the only commercial assay that uses this design. It is
a modification of the ouchterlony test, but instead of a gel matrix, it uses a two chamber,
L-shapted device. The vertical or motility chamber contains semisolid agar, and the top
of this chamber is inoculated with an antibody specific for salmonella flagellar antigen.
The horizontal or inoculation chamber contains selective enrichment and is inoculated
with an aliquot of the preenrichment broth. Motile salmonellae, if present, are growth-
amplified in the selective enrichment chamber and migrate into the motility chamber.
When the diffusing antibody contacts the flagellar antigen, a visible line of precipitation
is formed. Nonmotile salmonellae are not detected in this assay.

Enzyme-Linked Immunosorbent Assay (ELISA)

The ELISA is the earliest and probably the most prevalent format of antibody assay used
for pathogen detection in foods. Commercially available ELISAs are usually designed as
a “sandwich” assay, whereby antibody-coated solid matrices are used to capture the
antigen (bacteria or toxin) from enrichment cultures, and a second antibody conjugated
with an enzyme is added to form an antibody-antigen-conjugate ‘sandwich.” Enzymes
commonly used are alkaline phosphatase or horseradish peroxidase. A colorimetric
substrate is then added, which is cleaved by the enzyme to produce a colored product that
can be recorded visually or with a spectrophotometer. The detection sensitivity of ELISA
is usually about 104 to 105 CFU/ml for whole bacterial cells and a few nanograms per
milliliter or less for toxins or prteins; hence, cultural enrichment or extraction is needed
before detection. Some ELISAs habe been modified to include signal amplification to
improve sensitivity. One of these, the ELISA ELCA (enzyme-linked coagulation assay)
for Closttridium botulinum toxins, uses a coagulation-based amplification for detecting
bound toxin-antibody complexes. For some assays, there are programmed autoanalyzers
that perform ELISA procedures and reduce hands-on time. These automated ELISA
instruments are commercially available, and most still use the sandwich antibody format.
Some, such as the enzyme-linked fluorescence assay (ELFA), have been modified to use
a fluorgenic substrate to enhance sensitivity. After enrichment, automated systems can
perform an ELISA in 45 min, as compared with a manual ELISA, which requires a few
hours.

Immunomagnetic Separation

In addition to their application in detection assays, antibodies can also be used in food
analysis to selectively capture bacteria and shorten cultural enrichment time. In the
immunomagnetic separation (IMS) technology, specific antibodies coupled to magnetic
particles or beads are used to capture pathogens of interest from preenrichment media.
IMS is analogous to selective cultural enrichment, whereby the growth of other bacteria
is suppressed while the pathogen of interest is allowed to grow. The difference between
the two processes, however, is that in selective enrichment, chemical reagents or
antibiotics are used to select for the pathogens, while in IMS, pathogens are selectively
captured by antibodies. Since reagents can be harsh and may cause cell stress or injury,
IMS is a milder alternative for target enrichment; also, the elimination of selective
enrichment steps shortens sample analysis time. Bacteria purified by IMS can be further
tested by plating onto selective media or can be identified by serological, genetic, or other
methods. IMS has been used to select for lysteria sp., EHEC of the O157:H7 serotype,
Salmonella sp., and other pathogens from foods, as well as from environmental and
clinical samples.

Immunoprecipitation

Recently, assays using immunoprecipitation or immunochromatography have also been

introduced for detecting pathogens in foods. The assay is basically a sandwich antibody
test, but instead of enzyme conjugates, the detection antibodies are labeled colored latex
beads or colloidal gold. Most of these assays consist of small, disposable plastic devices
or dip-sticks, with a sample inoculation chamber, a test chamber and a control chamber
where the results are read. After enrichment, a 0.1-ml sample is added to the inoculation
chamber, where it is wicked across to the test and control chambers in a few minutes and
the results are generated. Conceptually, the device consists of a series of absorbent pads
that are saturated, in sequence, with latex-bound detection antibody (inoculation
chamber) and capture antibody (test chamber), both of which are specific for the target
antigen. In the control chamber there is another capture antibody, but it is specific not to
the antigen but rather to the detection antibody. Hence, as the sample wicks across the
pads, the antigen, if present, reacts first with the labeled detection antibody; this complex
is then bound by the captured antibody (test chamber) to form a visible band of
precipitation. As the excess complex diffuses across to the control chamber, it is retained
by the second capture antibody to form another line of precipitation. The control chamber
ensures that the sample has flowed across the entire device, so that the appearance of
precipitation bands in both the test and control chambers indicates a valid positive test.
Immunoprecipitation assays are extremely simple, requiring no washing or
manipulations, and can be completed within 10 min after cultural enrichment.

LIMITATIONS AND IMPACT

The number of rapid methods available for detecting bacterial pathogens and toxins in
foods is impressive. However, as discussed earlier, food analysis remains challenging
because food matrices interfere with microbiological methods, including rapid methods.
As a result, despite the great impact rapid methods have had on food testing, there also
limitations.

LIMITATIONS

Enrichment

By themselves, most rapid methods can be done in a few minutes to a few hours; hence,
they are more rapid than traditional microbiological procedures. But, in the analysis of
foods, the problems inherent in food testing do not allow the direct application of rapid
methods to testing for pathogens in foods. As a result, samples still need to be enriched in
growth media before analysis, this continued reliance on culturing is the time-limiting
step and somewhat diminishes the impact of rapid methods. Still, enrichment is a
limitation only in terms of assay speed. Enrichment also provides essential benefits, such
as dilution of the interfering effects of inhibitors, differentiation of viable from nonviable
cells, and repair of stressed or injured cells.

Confirmation

An ideal application for rapid method is for quick screening of foods for the presence of a
particular pathogen or toxin. AOAC International-approced rapid methods are mostly
designated for preliminary screening-negative results are regarded as definitive, but
positive results are considered presumptive and must be confirmed by standard
microbiological methods. Unlike culture tests, which almost always yield a viable isolate,
many rapid methods require cell lysis or heat denaturation to expose antigens or targets;
hence, the viable cells needed for confirmation can only be obtained by repeat analysis of
the original enrichment medium using culture techniques. Although confirmation extends
analysis time by several days, it may not be an imposing limitation, since most results
encountered in the analysis of food samples are negative; there are few positive results to
be confirmed.

Interference

Evaluation of rapid methods shows that some methods may perform better in some foods
than others. This may be attributed mostly to interference by normal biota and by food
components, some of which can be especially troublesome for the technologies used in
these assys. For example, ingredients in infant formula and intrinsic peroxidase in some
vegetables cause false reactions in ELISAs that use peroxidase conjugates. Simple
procedural variations, such as insufficient washing of the microtiter plate wells, can
produce variable results in ELISAs, especially if the food contains highly “sticky”
[Link], an ingredient in foods that ingibits an antibody assay by interfering
with antigen-antibody interactions may not affect a DNA-based test. Other components
can inhibit DNA hybridization or Taq polymerase but have no effect on antibodies. Since
method efficiencies may be food dependent, it is advisable to perform some comparative
studies to ensure that a particular assay will be effective in the analysis of that food type.

Gene Expression

DNA-based assays are highly specific, but the specificity is often dictated by short
oligonulceotides. Hence, a positive result, for instance with a probe or PCR primers
specific for a toxin gene, only indicates that bacteria with those gene sequences are
present and that the bacteria have the potential to be toxigenic. It does not indicate that
the cell is necessarily viable in the case of PCR, or that the gene is actually expressed and
that the toxin is made. Bacteria carry genetic sequences that are not expressed owing to
genetic mutations or physiological factors. Similarly, in cases of clostridial and
staphylococcal intoxication, in which illnesses are caused by the ingestion of performed
toxins, DNA probes and PCR assays can be used to detect the presence of cells, but they
of limited use in detecting the presence of performed toxins in foods.

Single-Target Design

Almost all rapid methods are designed to detect a single target, which makes them well
suited for use in quality control programs to screen foods for the presence of. But in
surveillance programs, a food may be tested simultaneously for the presence of several
pathogens. Likewise, in illness investigations, the food implicated may be suspected to
contain a particular pathogen based on clinical symptoms, but the actual pathogen is
unknown. In these situations, when multiple pathogen analyses may be needed, the
requirement for several single-target tests to analyze one food sample may make the
procedure complex and costly.

Overabundance of Assays

Currently, there are at least 30 different commercial assays for testing for Salmonella sp.
or EHEC O157:H7. such a large number of options can be confusing and overwhelming
to the user; more important, this has limited the ability of the scientific community to
effectively evaluate and validate these assays.

Impact on Regulations

The microbiological safety of foods is primary concern to public health, the food industry
and regulatory agencies have implemented programs to monitor food for the presence of
pathogens and microbial toxins. Many of the methods used in these compliance,
surveillance, and quality control programs are time-consuming microbiological methods.
Moreover, most of the food samples analyzed are negative for the presence of pathogens.
This is an ideal situation in which rapid methods can be used to quickly screen large
numbers of samples to eliminate those that are negative, thereby facilitating the
performance of these programs. Before implementation, however, rapid methods should
be carefully evaluated or, preferably, validated to ensure that they can effectively detect
the pathogen of interest in that food type. Another issue of concern is that, currently, a
positive result by a rapid method is regarded as presumptive and needs to be confirmed,
but negative results are accepted without confirmation. This creates situation in which
false-negative results are not detected unless other assays are used simultaneously during
analysis. False-negative reactions are of great concern in terms of food safety and have
serious implications, as they may result in foodborne infections.

As detection methods improve, levels of sensitivity also increase. While this may be good
news for the consumer, the greater detection sensitivity can also create interesting
challenges for the food industry and regulatory agencies. For example, current
specifications for Salmonella sp. in ready-to-eat foods are “zero tolerance” or “absence.”
Likewise, microbial toxins are not permitted in foods, as the presence of any amount of
these toxins is regarded as adulterating the food. But these “zero tolerance” limits are
flexible, because the sensitivity of the methods varies, and “absence” may really mean
“not detected.” The problem with increased detection sensitivity is that it may give rise to
situations in which foods previously analyzed by conventional methods and found to
conform with the requirement of “absence” may no longer meet the same specifications if
a more sensitive method is used. Such cases have already occurred; e.g., canned foods
contaminated with low levels of bacterial toxins were not detected by traditional methods
but were detected by rapid methods. In these situations, will food manufacturers have to
modify their processing facilities or upgrade their quality control programs to comply
with the greater test sensitivity? What is the health risk of the lower levels of pathogen
and toxin detected? Will there be a need to implement changes every time a more
sensitive method is introduced? Since guidelines are method dependent, will sterile foods
be the ultimate limit as methods get more and more sensitve?