Abstract Background The GENE-UP® E. coli O157:H7 2 (ECO 2) assay (Performance Tested MethodSM 121805) incorporates Fluorescence Resonance Energy Transfer hybridization probes into its proprietary PCR technology for the rapid detection of E. coli O157:H7 in select foods. Objective The purpose of this validation was to evaluate the method’s interlaboratory performance and submit the result to AOAC INTERNATIONAL for adoption as First Action Official MethodSM for the detection of E. coli O157:H7 in select foods. Method The GENE-UP® method was evaluated in a multi-laboratory study as part of the MicroVal validation process using unpaired test portions for one food matrix, raw milk cheese (Comté, 34% fat, 0.8% salt). The candidate method was compared to the ISO 16654:2001 reference method. Fourteen participants from 13 laboratories throughout the European Union participated. Three levels of contamination were evaluated: a non-inoculated control level (0 colony-forming units (CFU)/test portion), a low contamination level (∼5 CFU/test portion), and a high contamination level (∼10 CFU/test portion). Data from that study were analyzed according to the Probability of Detection (POD) statistical model as presented in the AOAC validation guidelines. The difference in laboratory POD (dLPODC) values with 95% confidence interval across collaborators was calculated for each level between the candidate and reference method results, and between the candidate presumptive and confirmed results. Results The dLPODC values with 95% confidence interval were; 0.00 (–0.04, 0.04), 0.27 (0.04, 0.49), and 0.17 (0.01, 0.33) for the non-inoculated, low and high contamination levels respectively. Conclusions The dLPODC results indicate a significant difference between the candidate method and the reference method for both the low and high contamination levels, with the candidate method producing higher recovery of the target organism at both levels. Highlights The GENE-UP E. coli O157:H7 assay provides industry with a rapid, accurate detection method for E. coli O157:H7 in a broad range of foods.
Activated protein C reduces thrombin generation by inactivating factors V and VIII in the presence of protein S. This prompted us to develop an assay which would allow specific exploration of this reaction. The total amount of thrombin formed in plasma after activation by tissue factor and phospholipids was reduced by adding thrombomodulin. This addition allowed protein C to be activated by endogenous thrombin. The inhibition of thrombin generation (ITG) due to protein C activation could be measured by comparing thrombin formation in the presence and in the absence of thrombomodulin. ITG increased with both protein C and protein S concentrations. Normal values of ITG expressed as a percentage were between 40 and 65% and were not influenced by age or sex. ITG increased in patients under heparin therapy, decreased in patients under oral anticoagulant therapy and was decreased in women using oral contraceptives. This method could be used for screening patients for protein C and protein S deficiencies.
Abstract Background: The GENE-UP®Cronobacter assay (Performance Tested MethodSM 081801) is a real-time PCR technology for the rapid detection of Cronobacter species in select foods and environmental surfaces. Objective: The purpose of this validation was to evaluate the method’s interlaboratory performance and submit the result to AOAC INTERNATIONAL for adoption as a First Action Official MethodSM for the detection of Cronobacter species in select foods and environmental surfaces. Method: The GENE-UP method was evaluated in a multilaboratory study as part of the AFNOR NF VALIDATION certification process (NF102) following ISO 16140-2:2016 using unpaired test portions for one food matrix, reconstituted infant formula containing probiotics. The candidate method was compared to the ISO 22964:2017 reference method. Sixteen participants from fifteen laboratories throughout the European Union participated. Three levels of contamination were evaluated: a noninoculated control level (0 CFU/target test portion), a low contamination level (approximately 2 CFU/target test portion), and a high contamination level (approximately 10 CFU/target test portion). Data from that study were analyzed according to the probability of detection (POD) statistical model as presented in the AOAC validation guidelines. The difference in laboratory POD (dLPODC) values with 95% confidence intervals across collaborators was calculated for each level between the candidate and reference method results and between the candidate presumptive and confirmed results. Results: The dLPODC values with 95% confidence intervals were 0.00 (–0.03, 0.03), –0.08 (–0.19, 0.02), and 0.00 (–0.03, 0.03) for the noninoculated, low, and high contamination levels, respectively. Conclusions: The dLPODC results indicate no significant difference between the candidate method and the reference method or between presumptive and confirmed results for all three levels of contamination. Highlights: The GENE-UP Cronobacter assay provides industry with a rapid, easy to use method for the rapid detection of Cronobacter in a wide range of products and environmental samples.
A monoclonal antibody (mAb) binding to protein C (PC) heavy chain but not to activated PC was found to inhibit PC activation by free thrombin, suggesting that epitope involved the activation site. Using a set of overlapping synthetic peptides, we confirmed that this mAb recognizes the sequence encompassing the thrombin cleavage site (165QVDPRLI(171)). Surprisingly, epitope was only accessible in the absence of calcium, half-maximal inhibition of mAb binding occurring at 100 microM Ca2+. Thus, our antibody provides direct evidence that conformation and/or accessibility of the activation site differ between the apo and Ca2+-stabilized conformers of PC.
The VIDAS Listeria monocytogenes Xpress (LMX) test is an enzyme-linked fluorescent immunoassay designed for use with the automated VIDAS or mini-VIDAS instruments for the specific detection of L. monocytogenes using a 26 h proprietary enrichment broth. The VIDAS LMX method was validated according to harmonized AOAC Research Institute (RI) and Official Methods of Analysis guidelines in both the AOAC Performance Tested Method (PTM) and GovVal programs. In the PTM comparison studies, the VIDAS LMX method was compared to the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook, the U.S. Food and Drug Administration Bacteriological Analytical Manual, and AOAC Official Methods. The comparative food studies consisted of two main parts: internal testing and AOAC independent laboratory testing, which included seven food matrixes (deli ham, processed cheese, vanilla ice cream, cooked shrimp, smoked white fish, frozen spinach, and peanut butter). As part of the AOAC R1 GovVal program, the VIDAS LMX method was compared to the Health Canada MFHPB-30 method for the detection of L. monocytogenes in five ready-to-eat (RTE) meats (hot dogs, deli turkey, deli ham, fermented sausage, and liver paté). Twenty replicates of each inoculation level and five uninoculated controls were evaluated in each study. The LMX method also included the use ofchromogenic media, chromID Ottaviani Agosti agar and chromID L. mono. agar, for confirmation of LMX presumptive results. In both the PTM and GovVal evaluations, there were no significant differences in the Chi-square values for the LMX method when compared to reference methods. The additional parameters tested in the PTM evaluation (inclusivity, exclusivity, ruggedness, stability, and lot-to-lot) satisfied the AOAC RI performance requirements. In both the PTM and GovVal validation studies, the VIDAS LMX method demonstrated reliability as a rapid qualitative method for next-day detection of L. monocytogenes in a variety of foods, including RTE meats.
Summary The performance of a new automated ELISA for a rapid, individual and quantitative measurement of plasma D-dimer (VIDAS D-dimer) has been evaluated. First, a study of 100 patients was performed in order to choose the best couple of antibodies in comparison with an already clinically validated ELISA. Then the results were certified in a prospective study including 195 consecutive patients suspected of pulmonary embolism (PE). For a cut-off level of 500 ng/ml VIDAS D-dimer showed a sensitivity of 100% (95% confidence interval 92-100), a specificity of 37.6%, a negative predictive value of 100% (95% CI 93.3-100) and a positive predictive value of 33.1%. During a 6 months’ follow-up no patient (95% CI 0-6.4) with D-dimer <500 ng/ml presented a new suspicion of venous thromboembolic disease. These results suggest that this rapid and single-dose ELISA provides a very useful tool for the clinician to exclude on a day-to-day basis the diagnosis of PE.
