Part of the foodborne pathogens pillar. Here the protagonist is not a bacterium: to understand how an RT-PCR signal is read, it helps to have first seen PCR controls and the Ct value.
Table of contents
A pathogen that does not grow in the laboratory
Almost everything known about microbiological food analysis starts from a simple idea: if the bacterium is in the sample, it is made to multiply on a culture medium and it is counted or detected. With enteric viruses that logic does not work. Viruses do not multiply in food: they need a host cell, and what there is in a batch of oysters, raspberries or lettuce are particles that arrived there and are waiting.
Norovirus and hepatitis A virus (HAV) are the two most relevant viruses in food safety. The 2008 FAO/WHO report on viruses in food describes them as the most important foodborne viruses, and notes that the foods involved tend to be minimally processed ones, such as bivalve molluscs and fresh produce, rather than industrialized ones. This article explains why detecting them is different from looking for Salmonella, how the ISO 15216 standards do it and what can and cannot be concluded from a positive result.
Why they cannot be cultured
A review on foodborne viruses published in 2018 in the International Journal of Food Microbiology (Bosch and colleagues) sums up the situation: despite numerous attempts with monolayers and three-dimensional structures of different cell lines, reproducible replication of human norovirus in vitro was not achieved. For HAV, wild strains are also not easily cultured; there is a laboratory-adapted strain (HM-175) used in research studies.
There has been progress. In 2016, a group described in the journal Science the replication of several human norovirus strains in stem cell-derived intestinal enteroids, with bile as a necessary factor. It is a valuable research tool for studying infectivity, but it is not a routine method that a food laboratory can apply to a batch.
The practical consequence is that plate counting, which remains the reference for many bacteria (see traditional microbiological culture), does not exist for these viruses. Nor is there an enrichment step that makes them grow. What is detected is their genetic material: RNA, by reverse transcription followed by real-time PCR (RT-qPCR).
How viruses reach food
Infected handlers
An infected person sheds large quantities of virus in feces, and in the case of norovirus also in vomit. According to the CDC, norovirus can continue to be transmitted for two weeks or more after the person feels better. HAV can be transmitted up to two weeks before symptoms appear, and even by people who never develop them. In an analysis of gastroenteritis outbreaks in the United States between 2009 and 2012, cited by the Bosch review, restaurants were the most frequent setting for norovirus outbreaks and in most the cause was attributed to infected handlers (70%).
Bivalve molluscs
Oysters, clams and mussels filter large volumes of water and concentrate the viruses it contains if it received sewage. They are often eaten raw or barely cooked. The CDC warns that anyone who eats raw shellfish is at risk of getting norovirus. The Bosch review adds that enteric viruses can persist for weeks or months in molluscs. For the bacterial context of this same group of foods, see Vibrio in shellfish and aquaculture.
Berries and vegetables
Soft fruits and leafy vegetables are eaten without cooking and can be contaminated by irrigation water, by hands during harvest or by processing. Freezing does not eliminate them: the same review reports a reduction of less than 1.2 log10 in strawberries, whole and puree, after 28 days of frozen storage. A documented example is the norovirus gastroenteritis outbreak of September and October 2012 in Germany, which affected about 11,000 people and whose vehicle was, according to epidemiological studies, a lot of frozen strawberries. Maunula and colleagues detected norovirus in 7 of 11 samples from that lot, with three different genotypes, which suggested contamination from sewage rather than from a single handler. The risk of fresh produce in general is developed in microbiology in fresh fruits and vegetables.
How often do these viruses appear in berries? A 2024 meta-analysis estimated a prevalence of norovirus and HAV of 2.12% (95% CI: 1.74-2.59%), with high heterogeneity between studies, so the authors ask that the figure be taken with caution. They also observed a decreasing trend over time and a higher prevalence in berries of African origin than in those from other continents.
Minimal doses and why they matter
The Bosch review cites estimates of the norovirus infectious dose in a range of 15 to 1,300 genome copies, or 1 to 10 viral particles. With such a low dose, a contamination that would be irrelevant for a bacterium that needs to multiply can be enough to cause illness. The CDC indicates that norovirus symptoms appear between 12 and 48 hours after exposure; in the case of hepatitis A, incubation runs from 2 to 7 weeks, which makes it hard to trace the responsible food.
ISO 15216: the reference method
The detection of HAV and norovirus in food is governed by the ISO 15216 series, "Microbiology of the food chain. Horizontal method for determination of hepatitis A virus and norovirus using real-time RT-PCR". It has two parts:
- ISO 15216-1:2017 is the quantification method. It applies to soft fruits, leaf, stem and bulb vegetables, bottled water, bivalve molluscs and surfaces. A validation study with 18 laboratories from 11 European countries, carried out between 2012 and 2014 on seven matrices, generated the limits of detection and quantification and the repeatability and reproducibility values that were incorporated into the 2017 edition (Lowther and colleagues).
- ISO 15216-2:2019 is the detection method, that is, presence or absence of the viral genome in the portion analyzed.
The general workflow has three stages. First, the viruses are released or eluted from the food and concentrated; the procedure depends on the matrix. Then, the viral RNA is extracted with guanidine thiocyanate and adsorbed onto silica. Finally, it is amplified and detected by RT-qPCR with specific probes for norovirus genogroups I and II and for HAV. The extraction part follows the same logic we explained in nucleic acid extraction, with an added difficulty: the starting material is very scarce and the matrix is rich in inhibitors.
The controls that make the result interpretable
The Bosch review explains that the method includes criteria designed to avoid false negatives and underestimation of the amount of virus, through two controls that are added to those of any PCR (see PCR controls):
- Process control. A control virus is added to the sample before processing it to measure the efficiency of the extraction. A 2025 study on frozen berries processed with ISO 15216:2017 mentions a minimum recovery threshold of 1%. Without this control, a negative cannot be distinguished from an extraction that recovered almost none of the virus.
- Inhibition control. A control RNA is added to the RT-qPCR reaction to assess whether the extract contains substances that slow amplification. The presence of inhibitors in the matrix is one of the difficulties noted for the sensitive detection of these viruses in food.
To these are added the positive control and the no-template control of any run. The reading of each curve follows the rules we described in Ct value in real-time PCR, and results close to the limit of detection are interpreted with the caution of limit of detection and uncertainty.
What a positive means
This is the central question and the one most often misunderstood. RT-qPCR detects viral RNA; it does not distinguish between infectious and non-infectious particles. A 2025 study quantified this in a model with murine norovirus, used as a surrogate for human norovirus, inoculated in frozen raspberries and processed with ISO 15216:2017. The limit of detection of cell culture was 3.1 log PFU/25 g, while RT-qPCR reached 1.0 log PFU/25 g (Ct of 36.7 ± 0.6): a difference of 2 log. The authors conclude that a substantial part of the genomes detected do not represent infectious particles, and that high Ct values, above 35, may not indicate viable virus. It is a laboratory model with a surrogate, not a rule for all foods, but it illustrates the gap.
For practice, this implies three things:
- A positive result means that the viral genome was detected in the portion analyzed. It does not prove that the food causes illness or that it has caused it.
- A negative result is valid only for the portion analyzed, and only if the process and inhibition controls were satisfactory. Viruses are distributed heterogeneously: in the German study, the choice of extraction method changed the detection rate, and the authors highlight the role of sampling.
- The decision to release, hold or withdraw a batch combines the laboratory result with epidemiological evidence, the origin of the product and the history of the production area or the supplier. It is the same idea as presumptive versus confirmed result, with the caveat that here there is no culture to confirm viability.
This limitation also explains why the molecular method for viruses is different from the live-cell RNA approaches we discuss in RNA vs. DNA: the viral RNA detected by ISO 15216 is genome present, not a signal of metabolic activity.
Surveillance in molluscs and relationship to outbreaks
The microbiological criteria applied today to bivalve molluscs are bacteriological. EFSA, in its 2011 scientific opinion on foodborne viruses, recommended introducing microbiological criteria for viruses in bivalve molluscs, unless they are labeled to be cooked before consumption, and stressed that controls should focus on preventing contamination rather than trying to remove it from the food.
In 2019, EFSA analyzed the European baseline survey on norovirus in oysters: 2,180 valid samples from production areas and 2,129 from dispatch centers, taken over two years. Prevalence was 34.5% (95% CI: 30.1-39.1%) in production areas and 10.8% (95% CI: 8.2-14.4%) in batches from dispatch centers, with marked seasonality (more contamination between November and April) and lower contamination in class A areas. Results exceeded the limit of quantification in less than 10% of the samples. EFSA concluded that the current bacteriological criteria could be complemented with a criterion for norovirus, although a limit close to or below the limit of quantification would be difficult to apply, and clarified that the study assessed analytical capacity and not health risk.
When there is an outbreak, the food laboratory is usually one piece alongside the epidemiological investigation. A detection in the food is most useful when it can be typed and compared with that of patients, as happened in Germany, where the genotype found in the strawberries also appeared in some of the sick. That comparison between isolates is the same principle we describe in genomic sequencing of outbreaks.
Prevention: what analysis does not replace
Virus testing in food is a verification and investigation tool, not the main control. EFSA recommends focusing controls on avoiding viral contamination, rather than trying to remove or inactivate viruses from already contaminated food. That points to staff hygiene and to the source of water and raw materials. The CDC, for its part, recommends careful handwashing, cooking shellfish well and washing fresh produce, and points to vaccination as the main hepatitis A prevention measure. These measures are integrated into good manufacturing practices and the HACCP plan.
On the kit offering: the TAAG catalog does not currently include kits for norovirus or hepatitis A. If your control program requires them, it is advisable to consult the TAAG technical team.
Conclusion
Norovirus and hepatitis A virus do not multiply in food nor are they cultured in the routine laboratory, so they are detected by their RNA using RT-qPCR, with the ISO 15216-1 (quantification) and ISO 15216-2 (detection) methods. The process and inhibition controls are what make it possible to interpret a negative, and a positive indicates genome detected, not demonstrated infectious virus. That is why the result is read together with epidemiology, sampling and prevention measures, which remain the main barrier against these viruses.
About TAAG
If your food safety program includes molluscs, berries or vegetables eaten fresh, consult the TAAG technical team on how the controls for bacterial pathogens fit in with the rest of your verification plan.
Frequently asked questions
Why can norovirus not be cultured like a bacterium?
Viruses need a host cell to replicate. For human norovirus, reproducible replication has not been achieved in the usual cell lines; in 2016 its replication in human intestinal enteroids was described, but it is a research tool and not a routine method for food. That is why its RNA is detected by RT-qPCR.
Which standards regulate the detection of norovirus and hepatitis A in food?
The ISO 15216 series: part 1 (2017) describes quantification by real-time RT-PCR and part 2 (2019) the detection of presence or absence. They apply to soft fruits, vegetables, bottled water, bivalve molluscs and surfaces.
Does a positive by RT-PCR mean that the food has infectious viruses?
Not necessarily. RT-qPCR detects viral RNA and does not distinguish infectious from non-infectious particles. In a model with murine norovirus in frozen raspberries, RT-qPCR was 2 log more sensitive than cell culture, which indicates that part of the genome detected does not correspond to infectious virus.
Which controls does the method for viruses in food need?
In addition to the controls of any PCR, ISO 15216 includes a process control, a virus added to the sample to measure the efficiency of the extraction, and an inhibition control, an RNA added to the reaction to detect substances that slow amplification.
How often is norovirus found in oysters?
In the European survey analyzed by EFSA in 2019, 34.5% of the samples from production areas and 10.8% of the batches from dispatch centers were positive, with more contamination between November and April. Less than 10% of the samples exceeded the limit of quantification.