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GB 4789.45-2023: National food safety standard - General rules for verification of microbial test methods
GB 4789.45-2023
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard – General Rules for
Verification of Microbial Test Methods
ISSUED ON. SEPTEMBER 6, 2023
IMPLEMENTED ON. SEPTEMBER 6, 2024
Issued by. National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
1 Scope... 3
2 Terms and Definitions... 3
3 General Requirements for Verification of Microbiological Test Methods... 4
3.1 Selection of performance parameters... 4
3.2 Selection of representative foods... 5
3.3 Requirements for verification samples... 5
3.4 Requirements for data processing... 7
4.Verification of Performance Parameters of Microbial Testing Methods... 7
4.1 Sensitivity... 7
4.2 Inclusiveness and exclusivity... 8
4.3 Accuracy... 8
Appendix A Food Sample Classification List for Verification of Microbiological Test
Methods... 10
Appendix B Sensitivity Calculation Method... 12
Appendix C Calculation Method of Intra-laboratory Verification Accuracy... 13
Appendix D Calculation Method of Interlaboratory Validation Accuracy... 15
National Food Safety Standard – General Rules for
Verification of Microbial Test Methods
1 Scope
This Standard specifies the general requirements for the verification of microbial test methods
for national food safety standards.
This Standard is applicable to the verification process during the development and revision
procedure microbial test methods for national food safety standard.
2 Terms and Definitions
2.1 Method verification
Test activities that provide objective and valid evidence to confirm that the performance
parameters of an analytical method meet the intended use of the method.
2.2 Qualitative methods
A method that tests a sample for the presence of a target microorganism.
2.3 Quantitative methods
A method that determines the number or concentration of target microorganisms in a sample.
2.4 Reference methods
The traditional culture methods of microorganisms that are recognized or widely accepted.
2.5 Sensitivity
The ability of the qualitative method to be validated to detect target microorganisms from a
sample.
2.6 Inclusiveness
The detection ability of the method to be verified for target microorganisms.
2.7 Exclusivity
The anti-interference ability of the method to be verified against non-target microorganisms.
2.8 Accuracy
The degree of consistency between the test results of the quantitative method and the true value
of a sample.
2.9 Specimen
The part that is derived from a sample, and is used for testing and representative of its
characteristics.
2.10 Partial detection level
When a qualitative method is used to test a set of parallel samples, the contamination level of
the sample is when the detection probability is between 25% and 75%.
2.11 50% limit of detection (LOD50)
The concentration of target microorganisms in the sample when the detection probability of the
qualitative method is 50%.
2.12 Relative limit of detection (RLOD)
If there is a reference method for verification, the ratio of the 50% detection limit of the method
to be verified and the reference method.
2.13 Paired/unpaired analysis
When two qualitative methods test a sample at the same time, if the bacterial enrichment
method in the first step is the same, the same specimen of such sample can be used for analysis;
this analysis is called "paired analysis". If the bacterial enrichment method in the first step is
different, then using different specimens of such sample for analysis; this analysis is called
"unpaired analysis".
3 General Requirements for Verification of Microbiological
Test Methods
3.1 Selection of performance parameters
According to the type of method to be verified, select appropriate performance parameters for
verification according to Table 1.
different food subcategories are isolated from the corresponding food subcategories as much as
possible. Strains isolated from other laboratories or purchased from strain preservation
institutions can also be used. All isolated strains shall be accurately identified and their sources
shall be known and traceable.
For the verification of methods with confirmatory procedures, only one target microorganism
needs to be selected for each food subcategory. For the verification of quantitative methods
without confirmatory procedures, different types of representative microorganisms shall be
selected for each food subcategory. For example, the method verification samples for total
bacterial count can choose Staphylococcus aureus (representing Gram-positive bacteria), large
intestine Escherichia (representing Gram-negative bacteria) and Bacillus subtilis (representing
spore-containing bacteria).
Cultivate the target microbial strain under appropriate conditions. If there are multiple target
microorganisms, they shall be cultured separately first and then mixed in equal amounts.
If the sample does not contain natural background flora, background flora can be added if
necessary. The contamination level of the background flora shall be at least 10 times higher
than the target microbial concentration; and appropriate counting methods can be used to
confirm that the contamination level meets the requirements.
3.3.2.2 Artificial contamination of samples
The culture is diluted with diluent to the appropriate concentration required to contaminate the
sample. If dilution does not effectively remove the medium from the culture, rinse with the
diluent and centrifuge 2 to 3 times to remove the culture medium before diluting.
When culture medium diluent is added to a sample, the volume of the diluent must not affect
the state of the sample. When the volume of the diluent may affect the sample state, the diluent
of appropriate concentration shall be made into freeze-dried powder. When the target
microorganism concentration in the freeze-dried powder cannot reach the concentration level
required to prepare low-contamination level samples, the freeze-dried powder is mixed evenly
with other freeze-dried powder without culture medium to dilute to the appropriate
concentration of the contaminated sample.
It should select samples containing background flora, but ensure that the target microorganisms
are not contained in the sample. Inoculate culture medium diluted to the appropriate
concentration (diluent or freeze-dried powder) into samples. The samples shall be mixed well
and stable. If the samples are not easily mixed evenly, each sample can be prepared separately;
the amount of each sample is reduced to the amount of the specimen; and the entire sample is
used for testing. If the samples used for unpaired analysis are not easy to mix evenly, different
specimens can be prepared separately and regarded as the same sample.
3.3.2.3 Determination of sample acceptance reference value
For verification of non-reference methods, an appropriate counting method can be used to
determine the concentration of the added target microorganisms; and take the concentration
converted to the concentration of the target microorganisms in the sample as the sample
acceptance reference value.
3.4 Requirements for data processing
The method development laboratory needs to conduct abnormal value inspection and statistical
analysis on all laboratory verification data. The method can use statistical methods such as
Grubbs inspection or technical analysis methods. The counting results shall be converted into
commonly used logarithmic values before subsequent calculations are performed.
4.Verification of Performance Parameters of Microbial
Testing Methods
4.1 Sensitivity
4.1.1 Verification method
When performing interlaboratory verification, at least 20 parallel samples shall be tested.
When performing interlaboratory verification, each verification laboratory tests 8 parallel
samples.
The sample contamination level shall be a partial detection level. If there is no reference method
during verification and the acceptance reference value of the sample is unknown, the sample
acceptance reference value must be determined according to the method specified in 3.3.2.3.
If there is no reference method during verification, use the method to be verified for testing. If
there is a reference method for verification, respectively use the reference method and the
method to be verified for testing. During the test, one blank control and one positive control
(the contamination level is more than 10 times the partial detection level) are set up for each
food subcategory.
In this Standard, sensitivity is expressed as 50% limit of detection (LOD50) or relative limit of
detection (RLOD). If there is no reference method during verification, calculate LOD50
according to B.1.If there is a reference method during verification, calculate RLOD according
to B.2.
4.1.2 Verification requirements
If there is no reference method during verification, the sensitivity of the method to be verified
is expressed as LOD50.The LOD50 of the qualitative method has no acceptance limit; and the
LOD50 acceptance limit of the MPN method is 5 CFU. If the LOD50 of the MPN method does
not exceed the acceptance limit, then its sensitivity meets the requirements.
If there is a reference method for verification, the sensitivity of the method to be verified is
expressed in RLOD. For paired analysis, the RLOD acceptance limit is 1.5; for unpaired
analysis, the RLOD acceptance limit is 2.5.If the RLOD does not exceed the acceptance limit,
the sensitivity of the method to be verified meets the requirements.
4.2 Inclusiveness and exclusivity
4.2.1 Verification method
The strains selected for inclusiveness and exclusivity tests shall reflect the diversity of
phenotypes, genotypes and/or serotypes of target microorganisms and non-target
microorganisms. The strains selected for inclusiveness testing shall cover the types of the target
microbial taxonomic unit and its lower taxonomic unit (if any). The strains selected for the
exclusiveness test shall cover closely related species at the same level outside the target
microbial taxonomic unit; and shall also include the dominant species commonly contained in
the sample (the number shall not exceed 1/3 of the total number of strains selected for the
exclusiveness test).
The inclusiveness test requires the selection of at least 30 strains of target microorganisms. For
the inclusiveness test of the Salmonella testing method, at least 50 strains of Salmonella of
different serotypes shall be selected, which shall cover the main Salmonella serotypes.
Exclusivity testing selects at least 30 strains of non-target microorganisms.
Directly test the pure culture using the method to be verified. The inoculum size of the pure
culture in each test shall be 102 CFU~103 CFU.
4.2.2 Verification requirements
If the number of test results of target microorganisms detected in the inclusiveness test is equal
to the number of selected strains, then the inclusiveness of the method to be verified meets the
requirements.
If the number of test results of the target microorganism not detected in the exclusivity test is
equal to the number of selected strains, the exclusivity of the method to be verified meets the
requirements.
4.3 Accuracy
4.3.1 Verification method
For each food subcategory, three concentration levels of low, medium, and high shall be
selected. The low level is about 10 times the theoretical detection limit; and the high level is at
least 100 times the limit. If there is no limit, the maximum contamination levels of health
indicator bacteria and pathogenic bacteria are set to approximately 107 CFU/g (mL) and 105
CFU/g (mL), respectively.