Download Challenges to Developing Real-Time Methods to Detect Pathogens

Challenges to Developing Real-Time
Methods to Detect Pathogens in Foods
Despite progress, real-time nucleic acid-based assays to detect
pathogens in foods are not yet suitable for routine use
Lee-Ann Jaykus
ven with improved methods for detecting pathogens in foods and environmental samples, microbiologists
so mandated often face a “needle-ina-haystack” challenge. How does one
detect small numbers of pathogens amid large
numbers of harmless background microflora in
a large and complex sample matrix? Traditional
pathogen detection methods rely on culture enrichment, selective and differential plating, and
additional biochemical and serological methods, making for analyses that may easily extend
several days. Over the years, more rapid methods have replaced plating steps with DNA hybridization or enzyme immunoassays. However,
even these methods detect at best 103-104 CFU/g
of target pathogens, meaning that culture enrichment steps are still necessary, as is confirmation for presumptively positive results. The
overall time savings is minimal.
However, enzyme-based nucleic acid amplification methods, including the polymerase chain
reaction (PCR) and nucleic acid sequence-based
amplification (NASBA), represent a significant
advance, one that has the potential to speed the
overall analysis by replacing culture enrichment
procedures with those that amplify specific nucleic acid sequences. Moreover, these new methods are highly specific and can be used to identify microorganisms that cannot be readily
cultured.
E
Important Technical Challenges in
Applying Rapid Methods to Food Samples
Despite these advantages, however, those hoping to routinely use nucleic acid amplification
methods for detecting pathogens in food and
environmental samples still face several techni-
cal challenges, including: (i) low levels of contaminating pathogens; (ii) high volumes or high
mass (ⱖ25 ml or grams) compared to amplification volumes (⬍10 ␮l); and (iii) residual matrix
components that inhibit enzymatic reactions.
Additional challenges include the need to confirm findings by time-consuming procedures
and satisfying industry and regulatory concerns
when nucleic acid sequences from nonviable
pathogens are detected.
Thus, researchers in this field often report that
PCR or NASBA detection limits prove no better
than 102-103 CFU/g of food product, which is
only slightly better than what they report using
ELISA and DNA hybridization methods. By and
large, culture enrichments are still necessary to
amplify targets before they can be detected using
nucleic acid amplification procedures.
Recent developments, referred to as real-time
nucleic acid amplification technologies, can further reduce overall test times by replacing timeconsuming postamplification electrophoresis or
hybridization methods with methods based on
fluorescence resonance energy transfer (FRET)
(Fig. 1). Of particular note are two commercial
systems—the TaqMan®, which is available from
Applied Biosystems in Foster City, Calif., (http:
//home.appliedbiosystems.com) and the NucliSens®, which is available from bioMerieux of
Durham, N.C. (http://www.biomerieux.com).
The TaqMan® assay capitalizes on the endogeonous 5⬘3 3⬘ exonuclease activity of Taq
DNA polymerase by including a dual fluorophore-labeled oligonucleotide probe during the
PCR amplification cycle. The fluorescence produced by the 5⬘ reporter dye ordinarily is
quenched by the 3⬘ quencher dye. However, if the
probe hybridizes to its complementary amplicon
Lee-Ann Jaykus is
an associate professor in the Departments of Food Science and
Microbiology, College of Agriculture
and Life Sciences,
North Carolina
State University,
Raleigh.
Volume 69, Number 7, 2003 / ASM News Y 341
FIGURE 1
Representative detection and confirmation methods for nucleic acid amplification. (A) Traditional agarose gel electrophoresis and Southern
hybridization; (B) Real-time PCR detection of the tdh gene using the TaqMan® assay in serial 10-fold dilutions of an overnight V.
parahaemolyticus culture (courtesy of Angelo DePaola, FDA Gulf Coast Seafood Laboratory); (C) Fluoroscein (FAM)-labeled molecular
beacon used in NASBA reaction for the assessment of enterotoxin gene expression in Bacillus cereus grown in skim milk in late log/early
stationary phase (courtesy of John McKillip, Louisiana Tech University).
“mate” during the PCR reactions, the 5⬘ nuclease
activity of Taq polymerase lops off the fluorescent
dye molecule, which fluoresces freely in the absence of a neighboring quencher molecule.
Meanwhile, the NucliSens® Basic Kit is based
on NASBA, a transcription-driven isothermal
RNA amplification method. bioMerieux recently
introduced its NucliSens® EasyQ system, which
combines NASBA with molecular beacons, which
consists of a dual fluorophore-labeled oligonucleotide probe that is incorporated into the amplification cocktail. The molecular beacon probe sequence is flanked by a hairpin stem that is formed
by two complementary (yet unrelated) arm sequences, forming a stem-and-loop structure. One
of the arms is labeled with a fluorescent moiety,
the other with a quencher that is kept in proximity
because of the complementarity of the arm sequences. The probe is added prior to NASBA, and
if hybridization to specific amplicons occurs during amplification, this proximity is disrupted, leaving the fluorophore free to fluoresce.
These assays give results in real time because
the amplicon can be detected and confirmed
342 Y ASM News / Volume 69, Number 7, 2003
while it is being amplified. In theory, these assay
designs could enable nucleic acid amplification
to replace culture enrichment, while the newly
generated amplicons that hybridize to fluorescent probes and are immediately detected could
replace otherwise time-consuming culture confirmation steps. Because these two steps are
combined, total testing time could be dropped
from days to hours (Fig. 2). However, despite
the promise of these real-time detection strategies, their routine use for detecting pathogens in
food and environmental matrices will remain
limited until scientists are able to adequately
address the needle-in-a-haystack dilemma.
Concentrating Pathogens Initially Can
Improve Overall Analysis
Separating, concentrating, and purifying foodborne pathogens from sample matrices before
undertaking nucleic acid amplification steps improves the overall analysis (Fig. 3). Such procedures are necessary when detecting viral agents
from foods because, unlike those bacterial
Hold the Shellfish
Lee-Ann Jaykus says emphatically
that she will not eat two foods,
raw shellfish and sprouts. Her selective abstinence is not surprising
because her research focuses on
microorganisms that contaminate
foods–and both these foods are
notorious for being contaminated
by nasty microorganisms. “I tell
my students that when you are
eating raw shellfish, you’re eating
poop and all,” she says, laughing.
She learned this as a graduate
student when she studied raw
shellfish as part of her doctoral
research. Her aversion to sprouts
came later, about six years ago,
when her own undergraduate students decided to study the microbiological quality of sprouts as
their class project. “I was amazed
when the results came in.” she
says. “When I saw the levels of
bacteria, I stopped eating them.
It’s very difficult to control bacterial contamination on seeds. The
way they are grown—in high moisture at body temperature— it’s like
putting bacteria in an incubator
with a bunch of food and letting
them go crazy.”
Nevertheless, Jaykus has great
faith in the safety of the U.S. food
supply, although she wishes there
were faster methods for detecting
problems when they crop up. She
became aware of the need for
faster tests during the mid-to late
1980s, a time of several high-profile food-poisoning outbreaks.
She was working in a lab in
Modesto, Calif., where her duties
included conducting tests for bac-
teria in food products. “It was just
taking too long,” she says, referring to the wait before results were ready. “The processors
wanted their results, and we
couldn’t turn it over fast enough.
“We really have to develop alternative sample processing methods,” she adds. “We need more
scientists to be working in that
area, and better technology to
deal with these issues. We are
working with a couple of different
technologies to try to concentrate
bacteria out of representative
food sample sizes, and also technologies to concentrate and purify
nucleic acids that would provide
evidence of contamination.”
Jaykus developed an interest in
food science during her undergraduate days at Purdue University in West Lafayette, Indiana,
where she began studying medical
technology. “I was a little worried
I was going to be bored,” she says.
“Somebody told me I should look
into food science because it was
all of these different disciplines
together, including biology, chemistry, and engineering. So I took
my first microbiology course and
loved it. I really liked that you
could see the test tubes turn different colors during experiments. It
was very definitive–if it turned a
color, you got what you wanted.
Now that I’m a Ph.D., I know that
it’s not always that straightforward.” What she likes today is the
ability to combine her formal training in food science with her love of
biology “and apply it in a very
pathogens that can be cultured, viruses are inert
in food matrices. Unfortunately, separating and
concentrating bacterial pathogens from foods
can prove difficult because, unlike many viruses,
bacterial cells are highly sensitive to agents such
practical matter. Everybody is interested because everybody eats.”
Jaykus grew up in Ridgefield,
Conn., the eldest of three girls.
Her father is a land surveyor, her
mother teaches the fifth grade.
She earned her B.S. and M.S. degrees in food science at Purdue
University and her Ph.D. at the
School of Public Health at the
University of North Carolina
(UNC) in Chapel Hill. She currently is an associate professor in
the food science department at
North Carolina State University
(NCSU) in Raleigh. She is married
to a pediatric oncologist who is a
professor at UNC Chapel Hill.
Between them, they have four
teenagers. Before taking a position at NCSU, Jaykus worked as a
quality control manager for FritoLay, Inc. and as the microbiology
division manager for Dairy and
Food Labs, Inc., in Modesto.
Jaykus good-naturedly fields
comments about her cooking and
eating habits. “My husband says I
cook meat to death,” she says.
“We eat a lot of baked Frito-Lay
products. It took a while to get
used to them, but once you do, it
becomes part of your diet. At 44,
with high cholesterol, I no longer
can eat that high-fat stuff anymore.” However, she adds, “Since
it was one of my guilty pleasures, I
have to say this: there is nothing
better than hot Fritos, or potato
chips, right out of the fryer.”
Marlene Cimons
Marlene Cimons is a freelance writer
in Bethesda, Md.
as organic solvents and detergents that are used
to remove matrix-associated interfering compounds.
Approaches for concentrating bacteria need
to address three issues that plague environmen-
Volume 69, Number 7, 2003 / ASM News Y 343
FIGURE 2
A comparison of traditional and real-time detection methods.
tal and food microbiologists, namely (i) how to
separate pathogens from sample particulates;
(ii) how to remove inhibitory compounds associated with the matrix; and (iii) how to reduce
the sample size and also recover nearly 100% of
the target organism(s). Depending upon the
needs of the analyst, methods may be designed
to concentrate either entire bacterial populations or only specific segments. Preferably, these
methods will not destroy viability, meaning subsequent culture methods will work if needed.
Options for Concentrating Bacteria
Methods for separating bacteria from a food
matrix and then concentrating them depend on
several chemical, physical, and biological principles (Table 1). In general, the goal is to take a
25–50-g sample and reduce its volume to less
than 1 ml, with high recovery of viable target
bacteria and full removal of matrix-associated
inhibitory compounds. During these procedures, attractive forces between bacterial cells
and matrix components are disrupted and
344 Y ASM News / Volume 69, Number 7, 2003
blocked from recurring, preferably without killing the bacteria. Enzymes, detergents, and
changes in pH or ionic conditions provide ways
to dissociate bacteria from such matrices, albeit
with mixed success.
Centrifugation is a commonly used physical
method to separate and concentrate microorganisms from complex sample matrices. Often,
samples are centrifuged at low speeds to sediment food particulates, leaving bacterial cells in
the supernatant fluid. Alternatively, samples are
centrifuged at higher forces to sediment bacterial cells, although other particles of equal or
greater density will sediment as well. Differential and density gradient centrifugation methods
also may be used to separate bacteria from complex food matrices such as meats. Centrifugation efficiencies can be improved if particle diameters are increased. One way involves
removing electrostatic charges (typically by
changing pH) to allow particles to adhere and
thus coagulate. Alternatively, adding small
amounts of high-molecular-weight, charged materials that bridge oppositely charged particles
enables loose aggregates to form, or
FIGURE 3
flocculate, and these may be readily removed by centrifugation.
Filtration is another important tool
for concentrating bacteria. Filtering
through cheesecloth, filter paper, or a
similar material can remove solid food
particles from samples, while the bacteria are retained in the filtrates. Alternatively, a food product homogenate can
be passed through a filter designed to
retain the microorganisms based on
their size and chemical properties with
subsequent disposal of the filtrate. Filter type, pore shape and dimension, and
the physical and chemical properties of
the microorganisms all contribute to
recovery efficiencies.
Bacteria also may be immobilized using various materials, including ion exchange resins, lectins, and metal hydroxides. Some of these agents, such as
metal hydroxides, can be used in floc
form, while others are adsorbed to
beads or affinity columns. After the
bacteria within a food sample become
Schematic of the concept and power of bacterial concentration prior to the application of
attached to a solid support and are subreal-time detection.
sequently separated from the food matrice, they are desorbed and then concentrated. Enzymes or changes in pH or ionic
When considered together, many of the bacstrength may be used to desorb bacteria from
terial concentration methods are complex, expensive, and can be applied only to relatively
these immobilizing materials, with various delow-volume samples. Another common theme is
grees of efficiency.
the need for initially treating samples to desorb
Immunomagnetic separation (IMS) is curbacteria from food matrices. Although achievrently a widely used biologically based bacterial
ing a 50- to 100-fold sample concentration with
concentration technique. IMS combines the use
recovery of 100% of the microorganisms and
of monoclonal antibodies with magnetic spheres
complete removal of all matrix-related inhibito select target cells from a mixed population.
tory compounds is desirable, this goal is difficult
After allowing the antibody to bind target bacto achieve with current technologies.
terial antigens within a food matrix, target cells
are separated from mixtures by exposing them
to a magnetic field. For instance, monosized
Additional Sample Concentration through
superparamagnetic polymer particles, known as
Nucleic Acid Extraction
™
Dynabeads , are available from Dynal Biotech
Effective nucleic acid extraction methods can
of Oslo, Norway (http://www.dynal.no). IMS
further reduce sample volumes and remove mahas proved an effective tool for isolating several
trix-associated inhibitors. Although nucleic acid
foodborne pathogens, including Listeria monoextraction kits have become commercially availcytogenes, Escherichia coli O157:H7, and Salable during the last five years, many are made
monella species. However, even when IMS prefor use on relatively uniform clinical samples.
cedes nucleic acid amplification steps, detection
3
These nucleic acid extraction methods typilimits are rarely better than 10 CFU/ml of the
cally
begin with either enzyme lysis or cell solutarget bacteria in a food homogenate, meaning
blization
using guanidinium isothiocyanate, folculture enrichment is still often required.
Volume 69, Number 7, 2003 / ASM News Y 345
lowed by cleanup steps using organic solvents,
suspended silica, affinity purification columns,
or proprietary compounds. Each additional step
adds time and complexity to the assay, and
usually reduces nucleic acid yield.
A further consideration is that many of these
kits, even those marketed for environmental matrices such as soil, are designed for samples of
only 0.1–1.0 g. This means that pathogens will
need to be concentrated prior to nucleic acid
extraction for those samples containing relatively small numbers of target pathogens. Nonetheless, a good nucleic acid extraction step can
reduce inhibitory substances and further reduce
sample volumes 10- to 100-fold. If preceded by
a pathogen concentration step, a combined concentration factor of 100-fold or more means
that a 25-g sample can be reduced to 250 ␮l or
less, a volume that is more suitable for typical
molecular detection approaches (Fig. 3).
Residual Matrix-Associated
Amplification Inhibitors
Even with the best concentration and purification schemes, residual matrix-associated inhibitors typically remain in final extracts. These
inhibitors either prevent amplification, resulting
in false-negative results, or else reduce its efficiency, resulting in poor detection limits. These
inhibitory effects sometimes are more pronounced when target template levels are particularly low, which is precisely when one needs
higher amplification efficiencies.
The list of potential matrix-associated inhibitors is nearly endless; few are well characterized,
and others remain unidentified. Usage of efficient and more robust enzymes helps to overcome some problems with inhibitors. Also,
some investigators add enhancement agents to
increase amplification efficiencies in the presence of matrix-associated inhibitors. For instance, bovine serum albumin (BSA) is particularly effective in enhancing the efficiency of
DNA amplification from extracts with iron-containing molecules such as hemoglobin or humic
acids, which typically are found in meat-containing foods and environmental water samples,
respectively. BSA presumably scavenges these
inhibitory compounds, preventing them from
binding to and inactivating Taq DNA polymerase. Dimethylsulfoxide (DMSO), dithiothreitol
346 Y ASM News / Volume 69, Number 7, 2003
(DTT), and betaine are among other commonly
used enhancement agents.
Choosing the Appropriate
Amplification Target
Nucleic acid amplification assays fail to differentiate live from dead cells. Culture enrichments
prior to PCR do not fully overcome this problem
because nucleic acids from dead pathogens may
be detected even after such enrichments. Although some researchers suggest that 16S ribosomal RNA would be a good alternative amplification target, this molecule is also relatively
stable and therefore not a completely reliable
indicator of cell viability.
Messenger RNA is considered a more promising target for amplification. However, to be a
reliable indicator of viability, the target mRNA
should be species or strain specific, have a brief
half-life, and be constitutively expressed, preferably at high copy number. Meeting all three of
these criteria is a challenge and, although some
studies are promising, target choice remains an
important consideration. For instance, investigators report 1,000-fold differences in assay
sensitivity when using different mRNA targets,
and transcription levels for one specific target
can change with cell physiological state. Also
problematic is the fact that very little work has
been done to adapt these RNA-based amplification methods to detecting pathogens in food and
environmental matrices.
Feasibility of Real-Time and Endpoint
Detection Approaches
Proponents of real-time nucleic acid amplifications cite two major advantages to this approach: an ability to detect products as they are
being amplified and the potential to design
quantitative assays. Although both prospects
appeal to food and environmental microbiologists, many food safety regulations stipulate detecting either the presence or absence of particular pathogens and are based on zero-tolerance
standards. These rules appear unlikely to change
in the near future.
Also, although detecting a signal while a particular nucleic acid is being amplified may speed
up an assay by an hour or so, real-time detection
might be less important than endpoint detection
in which the specific products are detected immediately following the termination of their amplification. Even so, for some pathogens, quantitative real-time assays may be applicable in the
future.
For instance, having semiquantitative assays
for pathogens such as Vibrio vulnificus and
Vibrio parahaemolyticus could improve the
management of shellfish-harvesting waters, thus
protecting public health while helping the seafood industry. Food and Drug Administration
(FDA) standards specify that V. parahaemolyticus levels remain below 10,000 CFU/g in readyto-eat seafoods. This microorganism can be detected directly in oyster mantle fluids (shell
liquor) at a limit of 400 CFU/ml using real-time
PCR, according to Andy DePaola of the FDA
Gulf Coast Seafood Laboratory, Dauphin Island, Ala. Moreover, with further concentration
and DNA purification, this detection sensitivity
may be increased by 10- to 100-fold with relative ease.
Nonetheless, there are difficult issues to address before the food industry adopts this technology for routine uses. First, this industry traditionally insisted on culturing microorganisms
to identify them and, thus, is wary of the reliability of molecular techniques. Second, the cost
of reagents used in real-time assays is high
(sometimes over $25 per test), while the instruments cost from $30,000 to $50,000. These
costs are prohibitive for all but the largest companies. Finally, the industry is not equipped to
hire staff that is trained to do such testing. The
methods simply have to become less expensive,
more user-friendly, and more robust.
Applications to Bioterrorism, Other
Considerations
In facing bioterrorism threats, food microbiologists are seeking to improve their ability to
identify microbial agents in foods as quickly as
possible. Much like natural microbial contaminants, such agents could be widely dispersed
among different foods in low concentrations,
posing the same needle-in-a-haystack challenges
that are faced when dealing with other food and
environmental samples. Efforts to harness realtime detection strategies and couple them with
microarray or DNA chip technologies could
help to meet these challenges.
Meanwhile, further research is critical. Specifically, research is needed to identify, develop,
and refine prototype methods for evaluating the
microbiological safety of foods, water, and the
environment (Table 2). These methods should
concentrate pathogens and remove matrix-associated inhibitors, and should be universal, simple, rapid, and inexpensive. They would thus
eliminate or reduce the need for culture enrichments, yielding analyses in less than one day
and, preferably, faster. They should also minimize the chance for false-positive results and be
available on a commercial and fully certified
basis. Meeting these goals will require extensive
comparative studies between these new technologies and current standard culture methods.
ACKNOWLEDGMENTS
This article is based on a talk presented at the 102nd ASM annual meeting. I thank Andy DePaola of the FDA Gulf Coast
Seafood Laboratory for the figure for real-time detection of V. parahaemolyticus using the TaqMan® assay (Fig. 1B) and John
McKillip of Louisiana Tech University, Ruston, for the figure for real-time detection of Bacillus cereus using NASBA/
molecular beacons (Fig. 1C). I also acknowledge Christina Moore, who prepared Fig. 1 and 2, and the USDA-CSREES
National Research Initiative for financial support.
This work represents paper number FSR 03-13 of the Journal Series of the Department of Food Science, North Carolina
State University, Raleigh NC 27695–7624. The mention of trademarked products in this paper does not imply any
endorsement by the North Carolina Agricultural Research Service or criticism of similar products that were not mentioned.
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Volume 69, Number 7, 2003 / ASM News Y 347