AOAC RI ERP Final Action Recommendations eBook

AOAC EXPERT REVIEW PANEL FOR MICROBIOLOGY FOR FOODS AND ENVIRONMENTAL SURFACES AOAC Expert Review Panel Final Action Feedback

Expert Review Panel Additional Required Information

I. The following documentation change was approved following review by Yi Chen. Procedure Clarification for the DuPont™ BAX® System Real-Time PCR Assay for Salmonella (PTM #081201 and OMA 2013.02) DuPont Nutrition & Health Diagnostics has conducted an internal investigation to determine the root cause of a customer-reported issue with higher rates of indeterminate results when using the DuPont™ BAX® System Real-Time PCR Assay for Salmonella . The results of this investigation revealed that the time interval between hydrating PCR tablets and loading samples into the BAX® System instrument may play a role in the occurrence of indeterminate results for this assay. Therefore, we believe it is important to clarify in the AOAC-approved protocols for this test that hydrated PCR tablets should be held in the PCR cooling block for 10 minutes before processing samples in order to more accurately convey the parameters followed during the original validation studies. During the investigation performed by the N&H Diagnostics Quality Assurance team, it was discovered that customers who reported a higher rate of indeterminate results utilize laboratory layouts that allow for the immediate loading of hydrated PCR tablets into the BAX® System instrument. Meanwhile, the N&H Diagnostics laboratories used to perform AOAC validation studies require the user to hydrate PCR tubes in a BSL-2 laboratory, then move to a separate instrument room to process samples. This movement – including lab coat removal, hand washing and travel to the instrument room – was determined to be approximately 5-10 minutes. To test the effect of this finding, the N&H Diagnostics Quality Assurance team attempted to replicate the customer issue of indeterminate results by processing a set of test samples with the BAX® System real- time Salmonella assay immediately after hydrating PCR tablets. The results of this small-scale study demonstrated that samples not subjected to the 10-minute hold time returned a higher rate of indeterminate results due to a high initial fluorescence (see Figure 1), while samples processed after the 10-minute hold did not display this high initial fluorescence (see Figure 2). Furthermore, when customers who had seen a significant number of indeterminate results were asked to perform a 10- minute hold between hydrating PCR tablets and loading samples into the BAX® System instrument to simulate this movement time, most reported their issue resolved. As a result of this investigation, we recommend that the current customer specification for hydrating PCR tablets – which states that samples must be loaded into the Q7 instrument within 30 minutes of hydrating PCR tablets – be further clarified within the AOAC-approved protocol to convey that samples must be loaded into the instrument 10-30 minutes after hydrating PCR tablets to account for the required hold time after tablet hydration.

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