Canadian Requirements for Determining the Equivalence of Food Microbiological Methods of Analysis
Date published: December 18, 2024
Overview
This document sets out the minimum requirements that an alternative method to a microbiological reference method set out in the Table of Microbiological Reference Methods for Food must fulfill to be considered equivalent. The regulated party must demonstrate through method validation that their alternative method meets the requirements before it can be used to verify the compliance with the microbiological criteria of food. The requirements in question are modelled on those presently set out in Volume 1: Official Methods for the Microbiological Analysis of Foods of the Compendium of Analytical Methods.
The Canadian Requirements for Determining the Equivalence of Food Microbiological Methods of Analysis should be used in conjunction with the Table of Microbiological Criteria for Food and the Table of Microbiological Reference Methods for Food.
This document is published and maintained by Health Canada and is incorporated by reference in Division 30 of Part B of the Food and Drug Regulations.
Who is this document for
- Food industry
- Other interested stakeholders
In this document
- Purpose
- Demonstration of equivalency
- Requirements for qualitative and quantitative methods of analysis
- Additional requirements specific to qualitative methods of analysis
- Additional requirements specific to quantitative methods of analysis
- Requirements for colony identification methods
Purpose
This document defines the performance parameters and sets out other requirements that must be met to determine that a method is equivalent to a microbiological reference method, for the purposes of Division 30 of Part B of the Food and Drug Regulations.
This document does not replace existing method validation guidelines from Health Canada for microbiological methods of analysis.
Demonstration of equivalency
To determine if an alternative method is equivalent to a reference method set out in column 2 of the Table of Microbiological Reference Methods for Food, that method must be validated using procedures that will provide sufficient evidence to evaluate the performance of the method against its respective microbiological reference method using performance parameters described in this document.
Requirements for qualitative and quantitative methods of analysis
Requirements for selectivity studies
Inclusivity study
The ability of the method to detect a wide variety of strains representing the target microorganism (inclusivity) must be demonstrated by:
- Testing at least 50 pure strains of the target microorganism, except for Salmonella methods for which testing at least 100 pure cultures of different serotypes of Salmonella is required.
- Selecting strains that cover the range of diversity of the target microorganism, such as biochemical characteristics, serotypes, phage types and geographical distribution.
Exclusivity study
The ability of the method to resist interference by cross-reactivity with non-target microorganisms likely to be found in the tested food (exclusivity) must be demonstrated by:
- Testing at least 30 pure strains of non-target microorganisms, including:
- strains that are suspected to cause interference;
- strains naturally occurring in food tested; and
- strains closely related to the target microorganism.
Selectivity studies (inclusivity and exclusivity) can be omitted for methods such as aerobic plate counts and total plate counts, where the level of natural microorganism of a food commodity is measured.
Relevant food
The ability of the method to detect target microorganisms in foods typical of the samples to be tested, including foods that may present a challenge to the performance of the method, must be demonstrated. A sufficient number of foods must be tested, to have a proper representation of the diversity of that food.
Relevant strains
The ability of the method to detect target microorganisms must be demonstrated using strains likely to be detected in the foods tested.
Relevant stress
The ability of the method to detect target microorganisms in a stressed state must be demonstrated only when the manufacturing processes or conditions of the food are likely to cause injury to the target microorganism.
Relevant equilibration
The ability of the method to detect target microorganisms must be demonstrated after the food (inoculated with the target microorganism in a stressed or unstressed state, as appropriate) has been stored at the normal storage temperature for that food, for a relevant period, as follows:
- Frozen food: at least 2 weeks at -18°C or less
- Refrigerated food: at least 48h at 2–8°C
- Food stored at room temperature: at least 2 weeks at 20–25°C
Additional requirements specific to qualitative methods of analysis
A qualitative method of analysis is a method whose result is either that the target microorganism is detected or not detected either directly or indirectly in a unit of mass or volume.
Inoculation levels
The ability of the method to detect target microorganisms must be demonstrated using two inoculation levels. The low level of inoculation must result in a fractional recovery of positive samples, which is as close as possible to 50% (acceptable range: 25–75%) and confirmed positive by the microbiological reference method. The high level of inoculation must result in all or nearly all samples testing positive.
Co-inoculation
When detection of the target microorganism may be impeded by the presence of non-target microorganisms, the ability of the method to detect target microorganisms in the presence of non-target microorganisms must be demonstrated.
Number of samples
The ability of the method to detect target microorganisms must be demonstrated using a sufficient number of contaminated samples for each food. The number of samples must provide high probability of detecting a difference in performance between the alternative method and the reference method.
Performance parameters
The method must meet or exceed the following performance parameters when compared to the corresponding microbiological reference method set out in column 2 of the Table of Microbiological Reference Methods for Food:
- Sensitivity ≥ 98%
- Specificity ≥ 90.4%
- False negative rate < 2.0%
- False positive rate ≤ 9.6%
- Efficacy ≥ 94%
- Level of detection (LOD) of the method must be comparable to or exceed the lower limit of detection of the reference method.
Additional requirements specific to quantitative methods of analysis
A quantitative method of analysis is a method where the result is the quantity of the target microorganism in a unit of mass or volume, measured either directly, or indirectly, for example through colour absorbance or impedance.
Performance parameter
The method must meet the following performance parameter when compared to the corresponding microbiological reference method set out in column 2 of the Table of Microbiological Reference Methods for Food:
- Strict equivalence between the alternative method and reference method covering the range of target microorganism concentrations set out in columns 5 and 6 of the Table of Microbiological Criteria for Food must be demonstrated based on recognized statistical approaches.
Requirements for colony identification methods
A colony identification method is a method used to confirm the identity of target microorganisms that have been isolated as pure colonies on agar media.
Requirements for selectivity studies
Inclusivity
The ability of the method to detect a wide variety of strains representing the target microorganism (inclusivity) must be demonstrated by:
- Testing at least 100 pure strains of the target microorganism, except for Salmonella methods for which testing at least 150 pure cultures of different serotypes of Salmonella is required.
- Selecting strains that cover the range of diversity of the target microorganism, such as biochemical characteristics, serotypes, phage types and geographical distribution.
Exclusivity
The ability of the method to resist interference by cross-reactivity with non-target microorganisms likely to be found in the tested food (exclusivity) must be demonstrated by:
- testing at least 50 pure strains of non-target microorganisms, including:
- strains that are suspected to cause interference;
- strains naturally occurring in food tested;
- strains closely related to the target microorganism.
Performance parameters
The method must meet or exceed the following performance parameters when compared to the corresponding microbiological reference method set out in column 2 of the Table of Microbiological Reference Methods for Food:
- False negative rate ≤ 1%
- False positive rate ≤ 2.0%