Pitfalls of Environmental
Testing for Listeria with PCR
Are PCR-based methods appropriate for the detection of Listeria spp. in
environmental surface samples?
It is standard practice within the industry for food processors to evaluate efforts to control and eliminate Listeria monocytogenes from finished
product by monitoring the presence of the genus Listeria within the processing facility. The recurring presence of any Listeria spp. may indicate ineffective sanitation practices that can lead to the niche growth and
establishment of L. monocytogenes. If established, there is a high risk that
product contamination may occur. The
strategy of environmental monitoring in
conjunction with quick and aggressive response to positive test results allows
processors to continually improve their operations and minimize any risk to finished
product. However, the success of these efforts rests heavily on the performance of
the chosen detection method.
The most common Listeria detection
methods include agar or film plating methods, antibody-based lateral flow or enzyme
immunoassays (EIA) and polymerase chain
reaction (PCR) methods. Of these different
technologies, PCR typically offers the highest degree of sensitivity and specificity.
However, not all PCR systems are capable
of dealing with the complexities of detecting Listeria from environmental samples. While generally better at preventing false positives from cross-reacting organisms, the DNA sequences
targeted by some PCR systems actually prevent them from detecting all
Listeria spp. To serve as a truly effective food safety diagnostic tool, a
Listeria detection method should be able to reliably detect all members of the
Listeria genus. Once identified, the necessary corrective actions can be initiated to address these specific potential problem areas through focused
cleaning, sanitation, and follow up monitoring efforts. As such, methods
that fail to detect one or more Listeria species are ultimately less effective
at controlling Listeria than those capable of detecting all species and may
actually undermine the organization’s efforts to reduce the risk of product
contamination.
counteracting these inhibitory compounds
that can otherwise lead to indeterminate
or false negative results. The standard approach is to dilute the sample through a
multi-step enrichment protocol and a
sample preparation process. While typically successful at preventing inhibition,
this type of dilution protocol can also reduce the method’s sensitivity by providing
as little as 1.25 µL of the enrichment for
PCR analysis. This may compromise its
ability to function as an effective food
safety diagnostic tool.
biocontrolsys.com
PCR Pitfalls
One of the often sited difficulties associated with the use of PCR for detection of foodborne pathogens is its susceptibility to inhibition. PCR can
be inhibited by ionic detergents, high concentrations of salt, complex polysaccharides, proteinases, etc. These inhibitors generally exert their effects
through direct interaction with the DNA itself or interference with the polymerase enzymes responsible for producing the DNA copies. Environmental
surface samples from processing facilities are likely to contain one or more
PCR inhibitors that originate from cleaning and sanitizing agents, food
product residues and even the buffers or media found in the sample collection device. As such, any PCR method must have a suitable mechanism for
Avoiding Those Pitfalls
BioControl Systems’ Assurance GDS™
for Listeria spp. applies microbiology and
genetic detection technologies that enable
the system to overcome many of the pitfalls
commonly associated with using PCR The
method includes an immunomagnetic separation (IMS) sample preparation procedure
that addresses the problems associated
with PCR inhibition without compromising
sensitivity. The procedure utilizes IMS technology to capture the Listeria present in the
sample enrichment, concentrate them onto
antibody-coated magnetic particles and
physically separate the Listeria from any
potential inhibitory compounds. Additionally, it provides the added benefit of reducing the level of Listeria required at the
conclusion of the enrichment process. As
such, the total enrichment time can be reduced to just 22 hours.
Beyond IMS, the system contains two
additional levels of specificity in the form
of DNA primers and probes. Because both
are comprised of highly specific single
strand DNA sequences complimentary to
the target DNA, their combined presence
ensures that only the target DNA sequences are amplified and detected. Because the sequences contained in the
primers and probes target highly conserved DNA sequences from specific
genes common to the entire Listeria
genus, all members of the genus can be
detected.
Historically, the pitfalls associated with
PCR may have outweighed its potential
benefits as a tool for environmental monitoring. However, recent advancements in
technology have enabled processors to
accurately and reliably detect the presence of Listeria spp. from environmental
surface samples. The ability to provide definitive positive and negative results for all
Listeria spp. in just 24 hours has made
PCR a truly practical and valuable component of any food safety program.
