IJCA - Volume 3 - Flipbook - Page 60
58 The International Journal of Conformity Assessment
the AOAC website. This will contain the list of
(food) items tested in the comparative study,
and a summary of the data. The AOAC® O昀케cial
Method of AnalysisSM 2016.01 (Neogen Molecular
Detection Assay 2 – Salmonella) was granted after
an inter-laboratory study of the method. This study
report is published in the Journal of AOAC and can
be accessed through the AOAC website7.
ISO 16140-2:2016 validation data
Neogen MDA2 SAL validation per the ISO 161402:2016 protocol was validated via NF VALIDATION
by AFNOR Certi昀椀cation. The AFNOR certi昀椀cate
3M 01/16-11/16 and summarized validation study
report can be found on the AFNOR Certi昀椀cation
website.
(Food) item for implementation veri昀椀cation. This
laboratory will need to select one (food) item tested
during the validation study that also belongs within
the scope of laboratory application of the user
laboratory. For implementation veri昀椀cation of this
method, the laboratory chose: raw ground chicken
breast.
(Food) items for (food) item veri昀椀cation. Only
one (food) category is tested within the laboratory
application, so the laboratory is only required to
choose one (food) item for the (food) item veri昀椀cation
study, as well as testing environmental sponges
with Letheen broth to verify the “Other” (non-food)
category under their laboratory application.
STEP 3: IMPLEMENTATION VERIFICATION USING
eLOD50 (EXAMPLE: PROTOCOL 1)
For qualitative methods, both implementation
and (food) item veri昀椀cation entail determining the
estimated LOD50. The Estimated LOD50 (eLOD50) is a
de昀椀nition unique to this standard. In microbiology, the
term LOD (“level of detection”) is utilized for qualitative
methods, based on replicate analyses at three distinct
inoculation levels of the target analyte in a matrix. The
veri昀椀cation study is termed eLOD50 because, although
the laboratory conducts a study with replicates at
different inoculation levels, it does not test enough
samples to ful昀椀ll the requirements for an actual LOD50,
as generated during method validation.
The standard offers three protocols to select from
for determining the eLOD50. For each protocol, the
laboratory will need to inoculate test portions.
Protocols 1 and 2 require the use of your own culture
for inoculation and require inoculation of test portions
at three levels: high, intermediate, and low. Protocol
3 allows for the use of a standardized reference
material—inoculating with a known concentration of
the target microorganism—and requires inoculation
of replicates at only a low level of inoculation.
Suppose the laboratory opts to follow Protocol 1. The
laboratory must initially determine what the LOD50
was for raw ground chicken breast in the validation
study in order to determine how to begin inoculation
of their (food) items. A culture of Salmonella is grown
overnight, serially diluted, and plated to determine
the concentration of the inoculum. This will help to
prepare to dilute to the required concentrations of
inoculation for each level (per the standard).
The standard instructs to inoculate to 9x the LOD50
determined in the validation study for the high
inoculation level, 3x the LOD50 for the intermediate
inoculation level, and 1x the LOD50 for the low
inoculation level. If an LOD50 wasn’t provided in the
validation study (or there isn’t one available to use for
veri昀椀cation), then the standard advises to assume an
LOD50 of 1 for the low inoculation level, which then is
used to determine all three inoculation levels.
After inoculating the test portions, they should
be analyzed according to the instructions for the
method to be veri昀椀ed, speci昀椀cally the Neogen
Molecular Detection Assay 2 – Salmonella in this
instance. Record the number of positive results at
each inoculation level and use the most probable
number (MPN) tables provided within the standard
to determine the multiplier applicable for the low
inoculation level. This multiplier is used to ascertain
the eLOD50 and assess whether the result aligns with
the Acceptability Limits as de昀椀ned in the standard.
Because there was no LOD50 listed in the Neogen
Molecular Detection Assay 2 – Salmonella AOAC OMA
validation report, the laboratory assumed an LOD50 of
1. To determine if they had met the Acceptability Limit
for veri昀椀cation of qualitative methods, the laboratory
looked within the standard to Table 16 (Acceptability
Limits for the veri昀椀cation of validated methods), which
says the eLOD50 should be ≤ 4x LOD50. ISO has an
Excel® -based program (workbook)7 available on the
ISO TC34/SC9 website8 to make calculations and to
determine whether your results meet Acceptability
Limits, as shown below in Figure 3. In the example
shown, the eLOD50 was determined to be 0.98 (the
program rounded the Observed eLOD50 of 0.98 to
1.0), which is ≤ 4x LOD50, and therefore met the
Acceptability Limit. This completed implementation
veri昀椀cation for this method.
