Pathogen Testing: Immunoassay vs. Molecular Methods

Microbes are ubiquitous in our world, and as such, the battle for a safe and sanitary food supply requires vigilance on the part of food producers and processors. Pathogenic bacteria, in particular, pose a risk to the health and safety of any consumer, and care should be taken to ensure the likes of Salmonella, Listeria, and E. coli O157 are appropriately screened for. With so many methods available, how do you decide which is most suitable for your production plant or product? To answer this question, we’ll need to look at the two most common categories of pathogen testing in greater detail: immunoassays and molecular-based testing.

Immunoassay

The Basics:

In the realm of microbiology, immunoassays target antigens (specialized proteins) that exist on the surface of bacteria. Each genus and species of bacteria has a somewhat unique “fingerprint” of antigens that have allowed the development of tests that can quickly identify their presence.  The most common immunoassay is the ELISA (Enzyme-Linked Immunosorbent Assay) and a close cousin is the ELFA (Enzyme-Linked Fluorescence Assay).¹ Both tests introduce an inoculum of sample to a well coated in receptors (antibodies) that correspond to the antigen of interest. If the targeted pathogen is present, it will bind to the well and remain there when the rest of the inoculum is washed away. A second round of antibodies is then added to the well, binding the pathogen again and attaching a color or fluorescent tag. The presence (or absence) of color or fluorescence indicates a detected or not detected result.²

The Pros:

As compared to traditional plating methods, immunoassays have a much quicker turnaround time, meaning you can receive a not detected sooner than the several days to a week cell culture requires.² The methods are also well established, having been around for decades. Their long history has produced a wealth of literature and validations that bring confidence to any client who might be requesting these particular methods be run.

The Cons:

Despite the longevity of immunoassays, these tests can have a higher incidence of false positives as compared to traditional plating.² In theory, antibodies should only react with antigens on the pathogen the test is meant to detect. In practice, however, this is not always true. If an organism has a protein complex similar to that on the pathogen of interest, it can cross-react and produce a positive result.² This “lock and key” model is only as good as the specificity of the antibodies used, and they have limits. Immunoassays also cannot utilize internal controls in every well during the run, meaning there is no feedback as to whether each individual reaction is happening properly.

Molecular

The Basics:

Molecular methods dig a bit deeper and go below the surface of the cell into the genetic material of the bacteria itself. Cells from the sample are lysed, and the DNA or RNA from this “soup” extracted. (We will look at DNA going forward, as this is the more common target material.) Through the use of enzymes and cyclical heating and cooling, the DNA helix is unbound and artificially designed sequences of DNA (primers) target, bind and amplify characteristic sequences of the target pathogen if present. Depending on the type of molecular method being used, these primers have fluorescent tags (much like those in immunoassays) and can indicate a detected or not detected result based on the reported fluorescence curve.

The Pros:

Like immunoassays, molecular methods are much faster than traditional plating. For example, real-time PCR, a common method for molecular detection can be run in as little as half an hour (not including enrichment time).³ Molecular methods also have higher levels of specificity, as relying on unique gene sequences to produce positive results leaves little room for matching & flagging errors.² Finally, molecular methods can utilize an internal control in each well. These controls have a unique fluorescence wavelength which allows the instrument to detect whether the test is working properly for each sample being run.⁴ If the internal control for a sample fails, the lab knows to retest. Without this control, there is potential for false negatives.

The Cons:

A common concern with PCR tests is their ability to detect dead cells. If raw materials have pathogenic cells present prior to processing, the remnants of these initial cells can still produce a positive result even if they are no longer living cells capable of causing illness.⁵ To rectify this, methods have been developed to eliminate dead cell DNA, such as including a DNAse step which effectively eliminates these loose DNA strands.⁶ Aside from detecting dead cells, a secondary criticism observed is that the methods themselves do not yet have the length and breadth of validations and studies that immunoassay methods do. Molecular technology, while powerful, is relatively new on the scene, having taken off decades after immunoassays.

Conclusions & Case Study

In light of the pros and cons noted above, it probably comes as no surprise that both molecular and immunoassay testing can be a valid method choice.  Though neither test is perfect, they are both faster alternatives to traditional plating and assuming the results come back not detected, your product will be on the way to consumers much faster than it would have before these methods were common practice.  What ultimately sets one test before the other, however, is cost-saving potential. Molecular methods, with their high specificity, internal controls, and quick turnaround times have this potential.

As mentioned above, the use of DNA as the target for molecular testing means less flagging errors, and therefore fewer false-positive results. When a molecular or immunoassay test produces a presumptive positive result, the sample then undergoes confirmation testing via a traditional plating method. This adds days of wait time and further expense on to your sample analysis. If it turns out that the plated test is not detected for the pathogen, we then know the initial presumptive result was a false positive, and the plating was an unnecessary expense. By using a molecular method rather than an immunoassay, our local lab has actually seen a 20% reduction in false presumptive positive Salmonella results when a large client switched from an immunoassay to a molecular method. While this type of result may vary from product to product, the potential cost savings that high-level specificity can bring, coupled with the peace of mind that internal controls offer, provide a winning combination well worth considering.

Sources

1. “Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations.” J. Woan-Fei Law et al. Frontiers in Microbiology. 12 January 2015.  https://doi.org/10.3389/fmicb.2014.00770. (https://www.frontiersin.org/articles/10.3389/fmicb.2014.00770/full).
2. “A Summary Profile of Pathogen Detection.” A. Crispin Philpott. Food Safety Magazine April/May 2009. https://www.foodsafetymagazine.com/magazine-archive1/aprilmay-2009/a-summary-profile-of-pathogen-detection-technologies/.
3. “A review on detection methods used for foodborne pathogens.” Priyanka, B et al. The Indian journal of medical research 144,3 (2016): 327-338. doi:10.4103/0971-5916.198677 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320838/).
4. “Practical Considerations in Design of Internal Amplification Controls for Diagnostic PCR Assays.” Hoorfar, B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner, P. Fach. Journal of Clinical Microbiology. May 2004, 42 (5) 1863-1868. DOI: 10.1128/JCM.42.5.1863-1868.2004. (https://jcm.asm.org/content/42/5/1863).
5. “A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything.” P. Kralik, M. Ricchi. Frontiers in Microbiology. 02 February 2017. https://doi.org/10.3389/fmicb.2017.00108. (https://www.frontiersin.org/articles/10.3389/fmicb.2017.00108/full).
6. “DNase I treated DNA-PCR based detection of food pathogens immobilized by metal hydroxides.” H. Thi Thu Do et al. World Journal of Microbiology and Biotechnology.August 2009, Volume 25, Issue 8, pp 1491–1495. https://link.springer.com/article/10.1007/s11274-009-0031-5.

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