Advantages / Disadvantages Traditional Methods Written by A. Crispin Philpott

TESTING: A Summary Profile of Pathogen Detection Technologies (April/May 2009)

Dating to the earliest days of microbiology, cell culture detection and recovery methods for pathogens have evolved in complexity, selectivity and effectiveness since the late 19th century. To the extent that they ideally produce a visible bacterial colony as their endpoint, cell culture methods have long been considered the gold standard of microbiology.

In brief, the method involves the suspension of a sample in solution; the preparation of an optimal bacterial growth medium with appropriate nutrients, pH level and metabolic indicators; the mixing or plating of the sample and media in a petri dish, tube or similar vessel; the incubation of the mixture at an ideal bacterial growth temperature for a prescribed period of time; and finally, a quantitative assessment of the presence or absence of countable bacterial colonies. Adjustments to any of the above method components—nutrients, pH level, indicators, incubation time or temperature—may be made to alter the selectivity and outcome of the assay. Fastidious bacterial targets, such as Listeria spp., may require multiple enrichments and platings in a variety of selective media to reduce competitors and enable recovery of the organism.

From the standpoint of speed, cell culture methods may require from several days to a week or more to generate confirmed and actionable test results. In terms of accuracy, many are considered official, confirmatory and/or gold standard procedures—that is, a visible colony of the target organism is produced. The ease-of-use of cell culture methods is arguable. A trained lab technician, exercising good laboratory practices (GLPs), is needed for the sample and media preparations, the multiple test manipulations and the interpretation of results.

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Advantages / Disadvantages PCR Technology written by A. Crispin Philpott

TESTING: A Summary Profile of Pathogen Detection Technologies (April/May 2009)

In 1983, while a chemist with Cetus Corporation, Nobel Prize laureate Kary Mullis perfected the polymerase chain reaction (PCR), ushering in a diagnostic revolution in molecular biology. PCR technology is based on the use of heat-resistant polymerase from (Thermophilus aquaticus Taq) for the exponential amplification of target DNA sequences that have been captured by primers specific to the target’s DNA.

In brief, the procedure involves the following four steps: denaturation, annealing, amplification and detection. Via high heat in a thermocycling instrument, the sample’s DNA is denatured, or split into single strands. The primer, with the target’s specific DNA sequence, anneals or binds to the single DNA strand if the target sequence is present during a cooling phase. The Taq polymerase causes exponential amplification or replication of the bound DNA sequence with additional thermocycling. The detection of the amplified target DNA can be achieved through various methods, including agarose gel electrophoresis and melting curve analysis of intercalating dyes. Single-cell detection is dependent upon sample enrichment to a target level of 104. Therefore, as with ELISA, the speed of PCR technology is determined by the target and duration of sample enrichment, which currently ranges from eight to 48 hours. PCR accuracy compares favorably with official cell culture methods and may arguably be better, given the technology’s highly specific DNA basis. A number of PCR-based offerings have received AOAC approval.

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Advantages / Disadvantages ELISA Technique written by A. Crispin Philpott

TESTING: A Summary Profile of Pathogen Detection Technologies (April/May 2009)

The award-winning work of Philip Perlmann and Eva Engvall on immunoassays in 1970 at the University of Stockholm enabled the emergence one decade later of commercially available, enzyme-linked immunosorbent assay (ELISA) technology for rapid foodborne pathogen detection. Based on antigen/antibody-binding affinity, the most common application of ELISA technology is the “sandwich assay.” Antibodies with high specificity to the target pathogen are anchored to a surface—for example, the sides of a micro-titer well. The food or environmental sample to be assayed is placed in solution and dispensed into the well. If the target antigen is present, it will bind to the affixed antibody. After the sample is rinsed away, the antigen bound to the fixed antibody remains. Antibodies that are conjugated with an enzyme are then added to the well. If the target antigen is present on the bound antibody, then the conjugated antibody will bind to that complex, forming an “antibody-antigen-antibody sandwich.” The unbound, conjugated antibody is rinsed away and a substrate is added to the well. If the enzyme linked to the second antibody is present, then a colorimetric or fluorescent reaction will occur. The intensity of this signal determines the presence or absence of the target in the original sample—a positive or negative test result.

The detection limit of the ELISA method is in the neighborhood of 104–106. In order to detect a single target cell, the sample must be enriched prior to screening, elevating the target to a detectable level. Depending on the target organism, enrichment procedures currently run from eight to 48 hours. Therefore, the speed of ELISA technology is determined by the target and the duration of the sample enrichment.

The technology’s accuracy compares favorably with official cell culture methods according to method approvals granted by the Association of Analytical Communities (AOAC INTERNATIONAL). The incidence of false-positive results may be marginally higher due to cross-reactivity with non-target organisms.

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