Have No Fear, qPCR Is Here: How qPCR can help identify food contamination

Foodborne disease affects almost 1 in 10 people around the world annually, and continuously presents a serious public health issue (9).

Food Contamination-Strawberries-Blueberries-Magnifying glass
Food Contamination is common and can be seen in a variety of forms and food products.

More than 200 diseases have evolved from consuming food contaminated by bacteria, viruses, parasites, and chemical substances, resulting in extensive increases in global disease and mortality rates (9). With this, foodborne pathogens cause a major strain on health-care systems; as these diseases induce a variety of different illnesses characterized by a multitude of symptoms including gastrointestinal, neurological, gynecological, and immunological (9,2).

But why is food contamination increasing?

New challenges, in addition to established food contamination hazards, only serve to compound and increase food contamination risks. Food is vulnerable to contamination at any point between farm and table—during production, processing, delivery, or preparation. Here are a few possible causes of contamination at each point in the chain (2):

  • Production: Infected animal biproducts, acquired toxins from predation and consumption of other sick animals, or pollutants of water, soil, and/or air.
  • Processing: Contaminated water for cleaning or ice. Germs on animals or on the production line.
  • Delivery: Bacterial growth due to uncontrolled temperatures or unclean mode of transport.
  • Preparation: Raw food contamination, cross-contamination, unclean work environments, or sick people near food.

Further emerging challenges include, more complex food movement, a consequence of changes in production and supply of imported food and international trade. This generates more contamination opportunities and transports infected products to other countries and consumers. Conjointly, changes in consumer preferences, and emerging bacteria, toxins, and antimicrobial resistance evolve, and are constantly changing the game for food contamination (1,9).

Hence, versatile tests that can identify foodborne illnesses in a rapid, versatile, and reliable way, are top priority.

Conventional identification of food pathogens:

Currently, conventional identification of foodborne pathogens is achieved through selective enrichment and culturing, followed by biochemical testing or MALDI-TOF (1). This process can take time, 2–3 days for preliminary identification and sometimes up to 7 days for pathogen species conformation (1,6,3), which raises several issues in food-based testing including (2,3,4):

  1. The shelf life of some foods is limited.
  2. Quantity of pathogens increases over time.
  3. Culturing can lead to the passing of antimicrobial resistant (AMR) genes.
  4. False negative results due to non-culturable pathogens (VBNC) that are actually viable.

Due to these issues, some culture-free methods have been explored and developed to be more rapid and with higher sensitivity; however, each has their own limitations.

A few alternative tests for food-based pathogens:

Multiplex PCR (mPCR) methods can be used for multiple gene targets which enable detection of several pathogens simultaneously. Negatively, expansion of mPCR methods is often necessary, as primer design is extremely important and AMR genes are routinely discovered, requiring more primer panels. Conventionally, agarose gel analysis is also required, which is time consuming and not easy to automate (1).

Metagenomic methods involve sequencing all the DNA in a sample through a shotgun method. This is difficult to apply to food, as it is not good for detecting specific pathogen strains (1).

Immunoassays use antibody-antigen interactions, relying on binding strength between them to determine sensitivity and specificity (6). These are limited to classifications above the genus line and are not a quantitative method, relying on real-time PCR (qPCR) for further quantification. (1)

So, just start with qPCR:

Currently qPCR assays are becoming widespread in food safety testing, as they are faster and more reliable than traditional methods. qPCR uses fluorescent signals from probes or dyes to determine food contamination based on real-time intensity (amount) of PCR amplicons produced in the reaction. This is a less time-consuming option than gel electrophoresis. Multiplex qPCR is also possible for detection of multiple foodborne pathogens and for assays that rely on internal amplification controls (6). Further, qPCR is safer for cross contamination because no manipulation of the sample is necessary after the initial qPCR run (5).

qPCR does have its own limitations, but scientists have taken steps to reduce these.

Suboptimal DNA purification is a major limitation in qPCR (and other culture-free methods above), especially in foods containing high fat, oil, or polysaccharide content (8). However, selection of a high-quality DNA purification method can remedy this limitation. Promega has developed a kit to combat this issue, the Maxwell® RSC PureFood Pathogen Kit, which can effectively isolate amplifiable pathogen DNA from a variety of food samples in as little as 40 minutes. The method has been shown to successfully isolate E. coli in strawberries, Salmonella enterica from raw shrimp and cold cut roast beef, and Listeria monocytogenes from soft cheese, amongst others (7).

Note: This kit, along with the Maxwell® RSC or the Maxwell® RSC 48 instruments, allows for efficient binding with prefilled cartridges, and avoids common problems with clogged tips and partial reagent transfers, allowing for efficient and high-quality DNA purification. Furthermore, Promega’s GoTaq® Enviro qPCR ready-to-go master mix has demonstrated success with DNA amplification in the presence of inhibitors and has shown compatibility with multiplex as well as fast or standard cycling methods, making it an advantageous option for food-based qPCR.

An additional limitation to qPCR is its inability to distinguish between alive and dead cells. So, even though a qPCR test may identify pathogenic bacteria, it could be devitalized (dead) and safe to consume (5,3). A solution to this problem? The use of fluorescent dyes, which would enter cells with compromised membrane integrity, aka dead cells, would become visible in a light-transparent matrix. This, however, would not work on samples with insufficient light transparency. Additional food for thought, as RNA has low stability and would be degraded within minutes in a dead cell, in exchange for the culturing methods there could be a standard characterization between cell viability and the nucleic acid species that could be adopted to use in qPCR (5).

Overall, qPCR could be the next step in rapid and reliable food contamination testing.  

Works Cited

  1. Bloomfield, Samuel J., et al. (2023) Determination and Quantification of Microbial Communities and Antimicrobial Resistance on Food through Host DNA-Depleted Metagenomics. Food Microbiology. 110, 104162.
  2. CDC. (2022) How Food Gets Contaminated – Food Safety. Centers for Disease Control and Prevention.
  3. Foddai, Antonio C. G., & Irene R. Grant. (2020) Methods for Detection of Viable Foodborne Pathogens: Current State-of-Art and Future Prospects. Applied Microbiology and Biotechnology. 104(10), 4281–4288.
  4. Gill, Alexander. (2017) The Importance of Bacterial Culture to Food Microbiology in the Age of Genomics. Frontiers in Microbiology. 8(777).
  5. Kralik, P., & Ricchi, M. (2017) A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything. Frontiers in Microbiology8(108).
  6. Law, Jodi Woan-Fei, et al. (2015) Rapid Methods for the Detection of Foodborne Bacterial Pathogens: Principles, Applications, Advantages and Limitations. Frontiers in Microbiology. 5(770).
  7. Teter, S. (Retrieved May 4, 2023) Purification of Bacterial DNA from Food Samples using the Maxwell RSC PureFood Pathogen Kit. Promega Notes
  8. Schrader, C., Schielke, A., Ellerbroek, L., & Johne, R. (2012) PCR inhibitors – occurrence, properties and removal. Journal of Applied Microbiology113(5), 1014–1026.
  9. World Health Organization. (2022) Food Safety. Who.int.
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Shannon earned her B.S. in Molecular and Cellular Biology, with a double minor in Chemistry and Psychology, and a Technical Writing Certification from the University of Wisconsin-La Crosse. As a member of the Digital Marketing Team, she values researching and analyzing scientific discoveries, and the importance of spreading this information to others. Depending on the season, you can find her either on the trails snowmobiling or on the lake kayaking.

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