Examples of Types of PTM Modification Validations

Table 1. Level 2 Method Modification Examples and Data Requirements  Method Modification  Data Requirement 

Matrix extensions up to four matrices,  depending on the number of matrices  included in the original validation claim.  For example:  Original claim 4 – level 2 modification  would be acceptable for only one matrix.  More than five claimed matrices require  an independent lab study.  Original claim 6 – level 2 modification for  the addition of 4 more matrices is  acceptable without an independent study.   Changes to non‐critical reagents  (lyophilization, Taq,), does not include  reagents that may affect  specificity/sensitivity such as new  antibodies or primer/probe sequences.  Enhancement of method workflow (non‐ critical steps)  Non‐reagent changes, such as changes to  the cartridge or increase in number of  wells  Changes in matrix sample sizes 

Matrix comparison study for all matrices. 

Matrix comparison study for all matrices and samples  sizes with the reference method samples size. 

Inclusivity/exclusivity and matrix comparison data for a  subset of claimed matrices. In some instances lot‐to‐lot,  stability and robustness required. 

Testing of 3 matrices inoculated post enrichment;  inclusivity/exclusivity; lot‐to‐lot studies.  

Dilution to extinction experiment. Matrix study 1‐3  matrices tested depending upon the overall matrix claim  for the method (matrices chosen by AOAC).  Inclusivity/exclusivity and matrix comparison data for a  subset of claimed matrices (matrices chosen by AOAC).  Where possible, the testing of frozen lysates from  previous validation on new instrument.  Inclusivity/exclusivity and matrix comparison data for a  subset of claimed matrices.  

New instrument, thermal cycler, PCR  platform or other 

Addition of DNA preparation methods 

Addition or update reference method 

Matrix comparison study for a subset of matrices. 

Expansion of the inclusivity claim within  the same target analyte species; for  example, in the case of expansion of  Listeria species claim.  Expansion of stability claim for reagents/

Inclusivity and exclusivity testing (blind‐randomized). 

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