New DNA Test Detects Listeria in Ice Cream and Reveals How It Survives Freezing

By Pooja Toshniwal PahariaReviewed by Susha Cheriyedath, M.Sc.Oct 21 2026

Scientists tested 500 ice cream samples and tracked bacterial survival and growth across freezing and fluctuating temperatures, raising important questions about contamination risks and the limits of conventional food safety testing.

Study: Development of propidium monoazide-droplet digital PCR for Listeria monocytogenes quantification and growth modeling in ice cream. Image Credit: Corona Borealis Studio / Shutterstock

In a recent study published in the journal npj science of food, researchers developed a propidium monoazide (PMA)-droplet digital polymerase chain reaction (ddPCR) assay to quantify potentially viable L. monocytogenes in ice cream samples and model its growth under different temperature conditions. The test distinguished L. monocytogenes from other bacteria and detected concentrations as low as 1.0 × 10² CFU/mL, while maintaining performance in samples containing other microbes. By providing quantitative results without requiring bacterial culture or standard curves, this approach could complement conventional food testing and help researchers assess how temperature changes affect pathogen growth.

L. monocytogenes is a foodborne pathogen found in various food products. Researchers have detected this pathogen in seafood, dairy products, ready-to-eat foods, and fresh vegetables and fruits. Consumption of contaminated food can cause listeriosis, a potentially severe illness affecting susceptible individuals, including children, pregnant women, immunocompromised individuals, and older adults. While traditional culture-based methods are usually accurate, they are often time-consuming and labor-intensive, warranting testing and evaluation of advanced detection methods.

About the study

In the present study, the researchers used PMA treatment and ddPCR to screen 500 commercial ice cream samples collected from supermarkets and farmers' markets in Shanghai, China, between May and September 2024, for potentially viable L. monocytogenes. They selected ice cream because it is a widely consumed frozen dessert that has been implicated in listeriosis outbreaks. Mathematical models, along with plate count results, characterized pathogen growth under isothermal conditions and fluctuating temperatures.

The team evaluated primer and probe specificity using three L. monocytogenes isolates, five isolates from other Listeria species, and seven bacterial strains belonging to different genera. The researchers investigated interference from other microbes, including Listeria innocua, Escherichia coli, Cronobacter sakazakii, Klebsiella pneumoniae, Listeria welshimeri, Bacillus cereus, and Staphylococcus aureus. These non-target strains were added at high concentrations exceeding 10⁶ CFU/mL for analysis.

The researchers used mathematical models to describe bacterial growth and predict how temperature changes affected the growth rate. Regression equations assessed correlations between the assay and plate counts, while biochemical tests coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed L. monocytogenes strains recovered from positive samples.

Results and discussion

The primers and probes designed to detect the hlyA gene, which encodes the virulence factor listeriolysin O, produced positive results only when L. monocytogenes genetic material was present, indicating high assay specificity. In validation experiments, the assay detected the pathogen at concentrations as low as 1.0 × 10² CFU/mL and maintained good quantitative performance in the presence of high concentrations of non-target bacteria. DNA copy numbers also declined as plate counts decreased, indicating a consistent relationship between the two measurement methods.

Among 500 samples, only two (0.4%) contained detectable L. monocytogenes, yielding 40.6 and 7.25 copies per μL, respectively. The researchers also recovered two isolates through enrichment culture and confirmed their identity using biochemical testing and MALDI-TOF MS. The assay showed consistent performance under the tested conditions, with strong correlations between molecular and plate-count measurements under constant temperatures from 5°C to 40°C and laboratory-simulated temperature fluctuations of 4–9°C, −10–15°C, and −20–25°C. The researchers also found that the selected mathematical models adequately represented the observed bacterial growth patterns. Higher temperatures accelerated bacterial growth, allowing the population to reach its maximum level sooner. The maximum specific growth rate (μmax) increased significantly with increasing incubation temperature, peaking at 35°C. However, absolute measurements differed between methods, with PMA-ddPCR generally producing higher counts during early growth and plate counts recording higher maximum bacterial concentrations.

In separate frozen-storage experiments, bacterial populations remained relatively stable for 240 days at temperatures between −5°C and −20°C, showing no substantial growth or decline under the tested frozen-storage conditions.

PMA enters bacterial cells with damaged membranes and binds to their DNA when exposed to light, preventing its amplification during PCR. This allows PMA-ddPCR to distinguish cells with intact membranes from those with compromised membranes, helping detect potentially viable L. monocytogenes without relying on culture. However, membrane integrity alone does not confirm that cells can reproduce or cause infection, and residual DNA from dead cells may still influence measurements.

The ddPCR method also divides the sample into numerous small droplets, allowing target DNA to be detected even at low concentrations. By separating the sample into individual droplets, ddPCR may limit interference from unrelated genetic material and reduce the impact of substances such as fat and protein that can disrupt PCR reactions. This could improve detection of the bacterium in ice cream, even when other food components interfere with the analysis. However, the study did not include dedicated inhibition controls, so interference from the ice cream matrix could not be fully excluded.

Conclusion and future directions

The findings suggest that PMA-ddPCR can quantify potentially viable Listeria monocytogenes cells in frozen desserts such as ice cream. It may complement culture-based methods and provide useful data for microbial growth modeling, although it should not replace traditional culture or enrichment methods. Differences between molecular and culture-based estimates of bacterial growth, particularly at low temperatures, need further investigation before the models can inform regulatory decisions. While freezing prevents the pathogen from growing under typical frozen-storage conditions, it can survive freezing for prolonged periods. Although pasteurization generally eliminates the pathogen from raw materials, contamination may occur during ingredient addition and during subsequent processing, freezing, hardening, or packaging. Poor temperature control during storage, transport, and distribution may also compromise food safety.

Future studies should validate the assay across different food matrices and account for matrix-specific factors that may affect detection accuracy. Further testing under real-world conditions is also needed to establish its practical value. Incorporating commercial time-temperature data collected throughout production, transportation, retail display, and consumer storage could improve cold-chain risk assessment and help identify stages where temperature excursions may increase bacterial growth risks.