Purpose: The purpose of this study is to evaluate performance characteristics of the EHEC qPCR screening assay for regulatory use.
Methods: The qPCR runs between August 2007 and September 2010 were collected for evaluation. Data were compiled along with additional information about each sample and sub-sample collected from FACTS and ORADSS. Descriptive statistics and further sub-analyses including Ct values for positive screens and internal amplification controls (IAC) were calculated by laboratory, by program, and by food types. The qPCR runs were interpreted using PCR positive and negative template controls and IAC as the indices to one of three potential qualitative outcomes: negative, cannot rule out (CRO), or invalid.
Results: One thousand and sixty three qPCR runs representing 2,163 samples (16,177 sub-samples) were collected. Twelve samples were confirmed as positive culturally among 300 target positive CROs. One CRO sub-sample was confirmed to contain E. coli O157:H7; however, the uidA target was not detected in the initial screening. Routine regulatory use of the qPCR screening assay for E. coli O157:H7 was capable of ruling out 82.3% of tested sub-samples as negative within 24 hours. The inconclusive CRO results (12.7%) may be caused by high generic E. coli background, reagent lot, human error, or possible matrix inhibition.
Significance: The data suggest that actions such as training, adequate DNA decontamination and modified procedure are recommended to improve the performance of EHEC qPCR assay.