Reviewed and Approved by: Alex Brandt, Ph.D, Chief Science Officer, Certified Group
One-Minute Summary
- The complete testing method includes the analyte, matrix, assay, and testing parameters.
- When all four factors match an existing validation, no additional validation study may be needed.
- Changes to the analyte, assay, or testing parameters may require full validation or, when appropriate, a mini-validation.
- Matrix similarity helps determine whether no additional study, method verification, or full validation is warranted.
“Can We Test This With That?”
Food manufacturers routinely ask third-party laboratories a deceptively simple question: Can we test this with that?
“This” might be a familiar product such as meat, cheese, leafy greens, milk powder, nuts, seafood, chocolate, or pet food. It might also be a less common matrix such as inulin fiber, shiitake mushroom sauce, dried citrus peels, or gochujang. “That” might be a reference method or rapid alternative method that was not specifically validated for the product, target organism, sample size, or testing conditions the manufacturer wants to use.
To answer the question, the laboratory must evaluate the complete testing method and determine whether full method validation, method verification, or another study is needed. A food microbiology method validation assessment begins with the complete method, not only the assay.
This discussion reflects the approach Certified Group follows for qualitative food microbiology testing methods, primarily pathogen methods, under U.S. validation guidelines. Other laboratories and users may follow different policies.
Method Validation for Food Microbiology Definitions
The terms “validation” and “verification” are sometimes used interchangeably, but they describe different levels of study.
Method validation is a structured study used to demonstrate that a candidate method is fit for its intended purpose. A full method validation following an AOAC Appendix J-style design compares the candidate method with a reference method. The study includes:
- Five high-inoculation samples
- 20 low-inoculation samples
- Five uninoculated samples
All samples are culturally confirmed. With the trial and error that may be required, the work can take three to six months.
Method verification is a more limited study performed with the candidate method. Certified Group’s approach uses six inoculated samples and six uninoculated samples, with cultural confirmation of all samples. A verification is usually completed within one month.
A mini-validation uses the same study setup as a method verification but is used in place of a full validation when appropriate. Certified Group requires client sign-off before proceeding with this approach.
A full validation is warranted in some situations, but less extensive studies can support method use in others. The decision begins by identifying exactly what is being tested and how.
How Certified Group Evaluates the Complete Testing Method
Certified Group evaluates four factors when determining whether a food microbiology method is fit for a specific testing request:
Analyte + matrix + assay + parameters = complete testing method
The analyte is the target microorganism, such as Salmonella, E. coli O157:H7, or Listeria monocytogenes.
The matrix is the product being tested, such as peanut butter, ground beef, or mozzarella cheese.
The assay is the detection technology, such as an enzyme-linked fluorescent assay, real-time PCR assay, or standard PCR assay.
The parameters are the conditions used to perform the test, including sample mass, incubation time, and growth medium.
The validation need depends on whether each factor is covered by an existing method and, when a factor differs, how significantly that change may affect the method. This food microbiology method validation framework helps the laboratory identify what additional study may be needed.
When All Four Factors Match
The simplest situation occurs when the analyte, matrix, assay, and parameters all match an existing validated method. Because previous validation work has already demonstrated that the method is fit for its intended purpose, no additional validation study is needed to adopt it at the laboratory.
For example, consider a request to test a 125 g frankfurter sample for Listeria monocytogenes using a PCR method with a turnaround time of less than 30 hours. If an existing real-time PCR method covers the analyte, matrix, sample mass, and other parameters, the laboratory can use that method after performing mock samples and completing a proficiency test. All factors are then documented in the test SOP.
The matrix must still match the validated application. A compositional change, such as the use of plant-based proteins in the frankfurter, changes the matrix and requires another evaluation.
What Happens When the Analyte or Assay Is Different?
Changing the analyte or assay generally requires a full method validation or, at minimum, a mini-validation.
Each assay can perform differently, including first- and second-generation versions of an assay. A method validated for one platform therefore cannot automatically be transferred to another.
| Change requested | Existing validated application | Requirement |
| Assay change | Salmonella in 375 g peanut butter using PCR; an ELFA assay is requested with the same parameters | Full method validation because the assay differs |
| Analyte change | Salmonella in 375 g garlic powder using PCR; Listeria spp. is requested | Full validation or at least a mini-validation when appropriate |
Certain situations may justify a less extensive study. One example is a change from Listeria monocytogenes to Listeria spp. The laboratory must evaluate the specific request rather than assuming that every analyte change requires the same study design.
What About When the Matrix Is Different?
When the analyte, assay, and parameters match but the matrix differs, the required work depends on how closely the new product aligns with a validated matrix under AOAC matrix-extension guidelines.
| Matrix comparison | AOAC matrix relationship | Requirement |
| Beef trim to lamb trim | Same subcategory, 1.B.3 | No additional validation activity |
| Flour to bread rolls | Same category and class; different groups, A.1 and A.3 | Method verification |
| Fresh leafy greens to frozen cranberries at pH 2.3–2.5 | Same category; different classes | Full method validation |
Food Microbiology Method Validation Requirements When Parameters Change
Sample mass is one of the most frequently changed testing parameters. Manufacturers increasingly request testing of larger sample masses, while many methods were validated for only 25 g samples.
Increasing the sample mass requires full method validation or, at minimum, a mini-validation.
| Existing validated application | Requested change | Requirement |
| Salmonella PCR in 100 g of pasteurized liquid egg | Increase to a 375 g sample and scale up the media volume | Full method validation or, at minimum, a mini-validation before use |
Other parameter changes include incubation time, incubation temperature, and growth medium. Because these changes can significantly alter method performance, Certified Group strongly recommends full method validation when they are made.
The Laboratory Must Be Able to Defend the Method
Third-party laboratories receive a diverse range of products and testing requests. A defined evaluation process helps determine whether an existing method is fit for the requested analyte, matrix, assay, and parameters.
Full method validation is necessary in certain situations. In others, method verification, matrix extension, or an appropriately applied mini-validation may provide the needed support.
While customers may wonder why additional time may be required before a method is onboarded, ultimately the laboratory must be able to defend its decision to use a specific method for a specific testing request. Maintaining clear documentation of both the decision-making process and the validation or verification work is critical. A documented food microbiology method validation decision provides the rationale for the selected approach.
Contact our team to talk about your food microbiology testing needs.
