Detection of Listeria spp. and L. monocytogenes in pooled test portion samples of processed dairy products.

Listeria monocytogenes is a major foodborne pathogen. Testing multiple portions of the same final product is often required to verify the effectiveness of a food safety management system. Therefore, it will be advantageous to the laboratories to combine these test portions and process as one sample. However, combining samples for analysis, i.e., pooling, can be done only if there is no negative impact on the result. The objective of this study was to validate pooling of test portions for the detection of L. monocytogenes and Listeria spp. in dairy products as no scientific evidence currently exists to support this practice. Six representative matrices, namely, pudding, yogurt, brie cheese, 2% milk, ice cream and infant formula were spiked separately with stressed L. monocytogenes and Listeria spp. in 25 g and pooled test portions (375 g/250 g/125 g). Two methods, namely, ISO-11290-1:1996 Amd1:2004 and a validated alternative method Rapid'L.Mono were used for sample testing. Performance of a method in pooled test portions was considered to be satisfactory if the relative limit of detection (RLOD50; LOD50 [pooled test portion]/LOD50 [25 g test portion]) and limit of detection (LOD50) obtained was ≤2.5 and 1 CFU or MPN, respectively. Results obtained from L. monocytogenes and Listeria spp. trials were given equal weightage to decide on the impact of pooling. Acceptable RLOD50 and LOD50 values were consistently obtained in L. monocytogenes and Listeria spp. inoculation experiments when test portions were pooled up to 125 g for all matrices tested with both methods. While there was a slight delay for the primary enrichment of the pooled test portions to reach the desired incubation temperature when compared to the 25 g test portions, it did not negatively impact the outcome when samples were pooled up to 125 g. Background organisms were in general present at low concentrations and did not seem to adversely impact the recovery of the target organism in 125 g samples. Thus, pooling of test portions to up to 125 g for the detection of L. monocytogenes and Listeria spp. by two culture methods in processed dairy products has been validated.

Validation of test portion pooling for Salmonella spp. detection in foods.

Pathogen monitoring programs play a crucial role in the verification of the effectiveness of implemented hygiene control measures. Sampling and testing procedures included in pathogen monitoring involve the analysis of multiple test portions where all samples must be negative for the presence of pathogens for a certain test portion size. Many food safety programs require increased testing due to the risks that a pathogen may be present. Analyzing more than one test portion could prove to be expensive and labor intensive. When more than one test portion for a specified food item is to be tested, the test portions could be combined to form a pooled test portion to reduce laboratory workload, costs of reagents and further confirmatory steps, but only when evidence is available that pooling does not affect on the number of false negative results for different matrices. This study has been performed to demonstrate the equivalence of test portion pooling for Salmonella detection with five different methods using cultural, ELISA and Real Time PCR technologies. Twenty-three (23) different food items including confectionary products, meal components, infant formula, pet food and powdered beverages were validated. Other complementary parameters like impact of minimum and maximum incubation time for pre-enrichment, temperature profile, pH and Salmonella concentration after the pre-enrichment and background flora have also been considered in the study. The results showed that pooling test portions up to 375g for Salmonella detection is valid for the methods that were tested. Relative level of detection (RLOD50) values for 22 of the food items tested were acceptable (i.e. lower than 2.5) when comparing the reference sample size (25g) against the alternative pooled sample size (375g), provided the enrichment broth was pre-warmed and maximum incubation time is respected.