NordVal International. c/o Norwegian Veterinary Institute PB 750 Sentrum, N-0106 Oslo, Norway RAPID L. mono

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1 NordVal International / NMKL c/o Norwegian Veterinary Institute PB 75 Sentrum, N-6 Oslo, Norway Issued for: RAPID L. mono NordVal No: First approval date: November 5 Extension date: December 5 Valid until: December 7 RAPID L. mono Manufactured and supplied by: Bio-Rad Laboratories, Blvd Raymond Poincare, 9 Marnes-la-Coquette, France fulfils the requirements of the NordVal validation protocol. The reference was EN ISO 9-(996/ amendment ): Food microbiology - Horizontal for the detection and enumeration of monocytogenes -- Part : Detection and Part : Enumeration. NordVal International has studied the enclosures to the application and evaluated the results obtained in the validations conducted by the expert laboratory I Institut Pasteur de Lille, France, in accordance to EN ISO 6. NordVal has concluded that it has been satisfactorily demonstrated that the requirements of the NordVal validation protocol are fulfilled for RAPID L. mono, there are no statistical differences in the performances of RAPID L. mono and the reference for the detection and the enumeration of monocytogenes and the detection of spp. in foods and environmental samples. Further, it was demonstrated that confirmation is not necessary. Yours sincerely Date: December 5 Sven Qvist NordVal International Hilde Skår Norli NMKL Secretary General

2 PRINCIPLE OF THE METHOD The principle of the RAPID L. Mono medium relies on the chromogenic detection of the monocytogenes phosphatidylinositol-specific phospholipase C (PIPLC) and on the inability of these species to metabolise xylose. After ± hours of incubation, monocytogoenes forms characteristics blue (pale blue, grey blue to dark blue) colonies without a yellow halo. Colonies formed by other species of are white, with or without a yellow halo. The particularity of ivanovii species, infrequently found in food matrices, should be noted: it presents blue-green colonies with a yellow halo (xylose positive character). This halo can appear after to 8 hours of incubation. The selective mixture in the medium allows the inhibition of most interfering flora (Gram- positive and Gram-negative bacteria, yeast and mould) Thus RAPID L mono detects monocytogenes in hours and other species in and 8 hours. FIELD OF APPLICATION The is applicable for the detection and the enumeration of monocytogenes and for the detection of other species in food and environmental samples. COMPARISON STUDY Relative accuracy, relative sensitivity, relative specificity and agreement between results, kappa Detection of monocytogenes after ± hours of incubation In and 6, 8 product samples were analysed: were naturally contaminated, 7 artificially contaminated and non-contaminated. All the samples were analysed once by the two s. The following results were obtained: Matrices *PA NA ND PD Sum Relative AC Relative SE Relative SP Kappa Meat products % 95.% 95.%.9 Fish products % % 98.%.98 Dairy products % 96.% 98.%.9 Vegetable products 7 77 % % %. Environment samples % 98.6% %.98 Total % 98.% 98.%.96 * PA = number of obtained results that are positive with both the alternative and the reference NA = number of obtained results that are negative with both the alternative and the reference. ND = number of obtained results that are negative with the alternative and positive with the reference (possible false negative) PD = number of obtained results that are positive with the alternative and negative with the reference (possible false positive) Relative AC = The relative accuracy; the degree of correspondence between the response obtained by the alternative and the reference. Relative SE = The relative sensitivity; the ability of the alternative to detect the analyte compared to the reference Relative SP = The relative specificity is the ability of the alternative not to detect the target microorganism when it is not detected by the reference. Kappa = The degree of agreement between the alternative and the reference, kappa of,8 or higher is considered to be very good agreement. For the response to monocytogenes alone, after hours (+/- hours) of incubation of the agar plates, the relative accuracy obtained is 98.%, the relative sensitivity is 98.% and the relative specificity is 98.%. Nine discordant results were obtained: supplemental positive results and 5 false negative results. The positive samples by the alternative

3 log log log being confirmed positive samples, the sensitivities and specificities were recalculated with respect to all of the positive results and are: - 98.% sensitivity for the alternative, - 98.% sensitivity for the reference. The agreement between the two s is very good (Kappa >.8) for all matrices. The sensitivity is satisfactory ( 95%) for all matrices, confirmation is not considered necessary. Enumeration of monocytotenes after ± hours of incubation In 5 tests were performed on 5 food product/strain combinations. The samples were analysed in duplicate by each of the two s. The following results were obtained: Product/ Matrix / Strain Reference Alternative Meat product Rillettes monocytogenes /b ,5,5,5 Meat (rillettes) Alternative,5,5 5 Dairy products Untreated Milk monocytogenes /b ,5,5,5 Dairy products (milk) Alternative,5 5 Vegetables Cabbage monocytogenes b ,5,5,5 Vegetables (cabbage) Alternative,5,5 5

4 log Product/ Matrix / Strain Reference Alternative Fish products Salmon monocytogenes /b ,5,5,5 Fish products (salmon) Alternative,5 5 Environmental samples Process water monocytogenes /b environmental sample (processed water) ,5,5,5,5,5 5 Sample no alternative The mean of the standard deviation,.9.9 The uncertainty of the, U.9.9 The results of the comparison study shows that the estimated measurement uncertainty is identical for both the reference and the alternative, U =.9. The diagrams show that all the mean values of the results are overlapping when taking the uncertainty of the into account. Hence there is no statistical difference between the s and both s perform satisfactorily - the precision is good. Detection of other than moncotygenes after 8 hours of incubation In 6, product samples were analysed: 99 were naturally contaminated, 87 artificially contaminated and non-contaminated. All the samples were analysed once by the two s. The following results were obtained: Matrices *PA NA ND PD Sum Relative AC Relative SE Relative SP Kappa Meat products % 97.6% %.97 Fish products % 96.9% 96.9%.9 Dairy products % 96.7% 96.%.9 Vegetable products % 9.7% %.9 Environment samples % 97.7% 98.%.96 Total % 96.% 97.9%.9 * PA = number of obtained results that are positive with both the alternative and the reference NA = number of obtained results that are negative with both the alternative and the reference.

5 ND = number of obtained results that are negative with the alternative and positive with the reference (possible false negative) PD = number of obtained results that are positive with the alternative and negative with the reference (possible false positive) Relative AC = The relative accuracy; the degree of correspondence between the response obtained by the alternative and the reference. Relative SE = The relative sensitivity; the ability of the alternative to detect the analyte compared to the reference Relative SP = The relative specificity is the ability of the alternative not to detect the target microorganism when it is not detected by the reference. Kappa = The degree of agreement between the alternative and the reference, kappa of,8 or higher is considered to be very good agreement. For the response to other than monocytogenes, the results demonstrate that the agar plates must be incubated for hours, and for additional hours in the case of absence of colonies or of weak growth. After 8 hours of incubation, the relative accuracy obtained is 97.%, the relative sensitivity is 96.% and the relative specificity is 97.9%. Twelve discordant results were obtained: 5 supplemental positive results and 7 false negative results. The positive samples by the alternative being confirmed positive samples, the sensitivities and specificities were recalculated with respect to all of the positive results and are: - 96.% sensitivity for the alternative, - 97.% sensitivity for the reference. The agreement between the two s is very good (Kappa >.8) for all matrices. The sensitivity is satisfactory ( 95%) for all matrices, confirmation is not considered necessary. Detection Level The different matrices have been analysed 6 times at different contamination levels by both s. The detection level was found to be - cfu in a sample of 5g or 5 ml for all matrices. Inclusivity /exclusivity Inclusivity: 5 strains of monocytogenes were detected out of the 5 tested. strains of other than monocytogenes were detected out of the tested. Exclusivity: The study conducted in 998 and 999 of 7 strains not belonging to the geneus monocotygtes did not detect presence of any cross-reaction, even with strains listed in the bibliography as having PLPLC activity: Bacillus cereus, Clostridium perfringens and Staphylococcus aureus. For the accuracy study performed in 6, some strains were identified because they produced colonies that looked similar to other than monocytogenes; these were Bacillus, Enterococcus faecium, Oekskovia xanthineolytica, Gardnerella vaginalis and Lactobacillus. However, these few strains have a different appearance from in the GRAM test. 5

6 log COLLABORATIVE STUDY DETECTION OF LISTERIA MONOCYTOGENES The collaborative study was conducted in 6. Number of participating laboratories: 5. Results from one laboratory were excluded The analyses were performed on samples of pasteurized milk, artificially contaminated with a strain of monocytogenes (L7), originating in raw-milk cheese) at the following three contamination levels: cfu/5 ml Low level, about the detection level, - cfu/5 ml High level, about times the detection level, - cfu/5 ml The laboratories analysed samples, 8 replicates for each level using both the alternative and the reference. The following results were obtained: Sensitivity: 96% Specificity: % Relative accuracy: % Kappa:,99 Thus, the collaborative study showed no statistical difference between the results obtained by the two s. ENUMERATION OF LISTERIA MONOCYTOGENES The collaborative study was conducted in 6. Number of participating laboratories: 5. Four laboratories were omitted, one due to late receipt and the three others because the temperatures of the samples were > 8 C Matrix: Pasteurized milk Strain: monocytogenes L7 No of samples: 8, levels (blind, low, medium, high), sample each level The diagram below shows the results for the low, medium and high level analysed with the reference (Serie) and the alternative (Serie ) respectively. results collaborative study,5,,5,,5 Serie Serie,,5, samples As expected the variation between the results is higher at the low level. 6

7 The precision is given in the table below: Alternative Level (log ) Repeatbility limit, r (log ) Reproducibility limit, R (log ) Repeatbility limit, r (log ) Reproducibility limit, R (log ) The repeatability limit is the value less than or equal to which the absolute difference between two tests results obtained under repeatability conditions is expected to be with a probability of 95%. The reproducibility limit is the value less than or equal to which the absolute difference between two test results obtained under reproducibility conditions is expected to be with The precision is satisfactory, even for low levels. For the lowest level, the precision is better for the reference than for the alternative. However, it is not statistical significant. CONCLUSION The studies have shown that there are no statistical differences in the performances of RAPID L. mono and the reference for the detection and the enumeration of monocytogenes and the detection of spp. in foods and environmental samples. Further, it has been demonstrated that confirmation is not necessary. 7

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