ERP Micro December 2019

bioMérieux GENE-UP® Listeria species 2 (LIS 2) AOAC ® Certification Number 121803

PRINCIPLE OF THE METHOD (1) The GENE-UP ™ Listeria species assay is used for the rapid and specific detection of Listeria species in enriched food and environmental samples. The GENE-UP ™ Listeria species assay is to be used with compatible PCR strip tubes in the GENE-UP ™ Thermocycler. Each reaction vial in the GENE-UP ™ Listeria species assay contains all of the necessary components for PCR, including sample-specific primers and probes and an internal amplification control. The GENE-UP ™ Listeria species assay detects fluorescence at several wavelengths (channels) to allow for multi-target detection in the same reaction vessel. The software automatically interprets the results for the internal amplification control and determines the sample result based on the outcome of the control. Both the assay for the sample and the internal amplification control utilize dual Fluorescence Resonance Energy Transfer (FRET) hybridization probes. The probes consist of two different short oligonucleotides that hybridize to an internal sequence of the amplified fragment during the annealing phase of the reaction cycle. FRET only occurs after the two probes come in close proximity from hybridizing to the template DNA. The resulting fluorescent signal from the FRET interaction, which forms a real time amplification curve, is how the amplified target is detected by the GENE-UP ™ Thermocycler. After the PCR cycling program finishes, the PCR product(s) are melted to determine the presence of the target DNA. The software uses both the real- time amplification curve and the melt peak to make a positive or negative call. [5] Altogether, Listeria LT FSS results were equivalent to those for reference methods at low inoculum levels with individual and pooled samples. Out of 120 samples overall, evaluated in the sponsor laboratory, 64 samples were positive with the Listeria LT and 67 were positive with the appropriate reference method. Relative sensitivity overall was 96% for the Listeria LT FSS with individual samples and 93% with pooled samples. In sponsor laboratory results, the false positive rate was 0%, and false negative rate was 0% for individual samples and 3% for pooled samples, using the Listeria LT confirmation method for comparison. Results from the independent laboratory suggest that the Listeria LT method is better than the reference method for turkey deli meat. Chi square values ranged from 18-23 indicating a statistically significant difference between the reference method and the Listeria LT method. This difference is due to a lack of Listeria detection with the reference method at such a low inoculation level. Independent turkey testing occurred at inoculation levels of 0.38 MPN/25g, compared to the sponsor lab testing which was performed at ten-fold higher levels (3.75 MPN/25g). Both tests returned fractional inoculation results. It is possible that the Listeria LT is better at detecting very low levels of Listeria equilibrated in deli turkey meat than the reference method in some circumstances. It is also possible that sampling error occurred due to such a low MPN. The independent laboratory confirmed that all controls gave expected results and there were no problems with the reference method. This difference was not observed during development or in the sponsor laboratory testing, which was conducted in an external laboratory. Because development was aimed at optimizing recovery of Listeria , rather than matching the reference method, it is possible that the Listeria LT FSS protocol generates more sensitive results than the reference method. The Listeria LT method for food samples uses Oxoid ONE broth, which may present an advantage to stressed Listeria cells, and might improve recovery over reference method medias. Although the false negatives for the test kit seem high in this case, in comparison to the reference method, the test kit was more sensitive. Results from the independent laboratory verified that the Listeria LT method is equivalent to the appropriate reference method for stainless steel environmental surfaces. The false positive and false negative rates were higher than observed at the sponsor laboratory (Marshfield Food Safety Laboratory) during the AOAC evaluation and at Idaho Technology during development. One presumptive positive sample was confirmed negative and a presumptive negative sample was confirmed positive, which led to the 8% false negative and the 13% false positive numbers (see Table VI). It is possible that samples were mixed up, since the number of positive samples and confirmed samples were the same. It seems unlikely that the test would both miss a low level sample, and give a false positive. Also, a less than ideal confirmation procedure was followed because ITI failed to include the confirmation protocol initially. Initially, Silliker began the confirmation at the same time as the Listeria LT PCR sample was tested (after only 24 hours of enrichment). The confirmations were more consistent when the sample was allowed to incubate for a total of 48 hours before confirming. ITI resubmitted protocols to include the confirmation procedure before turkey was tested. One uninoculated control sample, for ceramic surfaces, was initially called positive. There was clearly no actual amplification in the real-time fluorescence signal. This sample was repeated and found to be negative. Therefore there was no background Listeria in those samples. The cause of the positive call was determined to be a bump in the signal that was incorrectly interpreted as a positive by the detector software. Since the cause is known, a fix for this incorrect call will be included in the The Listeria LT FSS is highly specific and was able to detect 54 out of 54 strains tested in the inclusivity panel. The 54 strains were taken from all five target species, including 16 different serotypes. It did not detect 35 out of the 35 strains tested in the exclusivity panel, comprised of 31 non- Listeria species and Listeria grayii . Each of the 54 Listeria strains in the Inclusivity panel, was tested in two ways: grown in BLEB or grown in Oxoid ONE broth with supplements, without any food present. All 54 were detected when grown in BLEB. Only 49 were initially positive in ONE after 24 hour enrichment without food present. The remaining five were positive in ONE if incubated for 48 hours, or if incubated 24-28 hours with food was present. This is consistent with preliminary development data in our lab, which suggested that ONE broth does not support the growth of some Listeria strains very well without food present. One of the strains that could only be recovered after 48 hours in the ONE broth, Listeria selligeri is an avirulent strain (ATCC 355967), was also used for the method comparison study on the ceramic surface. The additional strain that required 48 hours in the ONE broth ATCC 19111 was a 1/2 a strain. Three additional 1/2a strains were detected if incubated 24-28 hours with food present. Ruggedness and Reagent Variation: The Listeria LT FSS is robust and reproducible as demonstrated by the ruggedness, lot-to-lot and shelf life studies. The ruggedness study demonstrated that the system produced consistent results with user introduced variability in reagent preparation time and sample volumes. The lot to lot and shelf life study demonstrated that the Listeria LT FSS gave consistent results with several lots of reagents produced at different times, including one lot close to the age of expiration. next version of software. Inclusivity and Exclusivity: DISCUSSION OF THE ORIGINAL VALIDATION STUDY (1) Comparison to Reference Methods: