Validation of the AuroFlow™ PR1ME™ BT Combo MRL Assay for the detection of beta-lactams and tetracyclines in raw commingled cow’s milk.

General Information

AuroFlow™ PR1ME™ BT Combo MRL Assay is a competitive assay in a test strip format for the rapid detection of residues of beta-lactams plus metabolites, including penicillins, cefalosporins, and tetracyclines in raw commingled cow’s milk. The assay detects 17 beta-lactam (including select metabolites), and 3 tetracyclines below European MRL levels, and can obtain results in 7 minutes.

Principle of Method

A raw milk sample at 2-15°C is mixed to ensure sample homogeneity. An empty tube is placed into the AuroBLOCK™ incubator. 200 µL of milk is added to the tube, followed by the test strip with the arrows pointing down. After an incubation for 7 minutes at 50°C, the test strip is removed from the tube and the results are interpreted visually by comparing the intensities of the beta-lactam, ceftiofur, and tetracycline lines with the intensity of the control line, or by reading the strip in the QuickSTAR™ Strip Test Reader and applying a ratio of 1.00 as the cut-off value. If the antibiotic test line is less intense than the control line, the sample is positive for that antibiotic.

Ratio (area measurement) Interpretation

≥ 1.00 Negative

< 1.00 Positive Table 1. Instrumental reading: interpretation of test results.

AuroFlow™ PR1ME™ BT Combo MRL Assay Catalog #FOOD-1134-02

Author

Nicolas Kosa PerkinElmer, Inc. 7050 Burleson Road Austin, TX 78744

Contributor

Leo Mendoza PerkinElmer, Inc. 7050 Burleson Road Austin, TX 78744

Contributor

John Moore PerkinElmer, Inc. 7050 Burleson Road Austin, TX 78744

Contributor

Marit Terweij PerkinElmer, Inc. 7050 Burleson Road

Austin, TX 78744

Contributor

Madhavi Vemuganti PerkinElmer, Inc. 7050 Burleson Road

Austin, TX 78744

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Figure 1. Visual Interpretation of the AuroFlow™ PR1ME™ BT Combo MRL Assay test strip.

The predicted detection capability (CCβ) for 15 beta-lactams and 4 tetracycline antibiotics was determined with the AuroFlow™ PR1ME™ BT Combo MRL Assay. Antibiotic free milk samples were fortified with each antibiotic at a range of concentrations at, above, and below the Maximum Residue Limit (MRL). Each sample was tested with 10-20 test strips per concentration from a single lot of test strips. The detection capability was defined as the lowest analyte concentration of a single milk sample tested yielding positive results for at least 19 out of 20 strips tested following the Guidelines for Validation of Screening Methods of Veterinary Medicines (Community Reference Laboratories 20/01/2010). 115 antibiotic milk samples were also tested.

Antibiotic EU MRL (µg/kg)

Concentrations Tested (µg/kg)

Amoxicillin 4 1, 2, 3, 4

Ampicillin 4 1, 2, 3, 4

Cefacetrile 125

Cefalonium 20 4, 6, 8, 20

Cefazolin 50 25, 50, 60, 75

Cefoperazone 50 1, 1.5, 2, 3

Cefquinome 20 5, 10, 15, 20

Ceftiofur 100 10, 20, 30, 100

Cefalexin 60 100, 500, 1000, 1500

Cephapirin 60 4, 5, 6, 60

Cloxacillin 30 2, 3, 4, 30

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Dicloxacillin 30 1, 2, 3, 30

Nafcillin 30 2, 4, 6, 30

Oxacillin 30 2, 3, 4, 30

Desfuroylceftiofur 100 10, 20, 30, 100

Penicillin G 4 1, 2, 3, 4

Desacetylcephapirin 60 10, 15, 20, 60

Tetracycline 100 5, 10, 20, 100

Oxytetracycline 100 10, 20, 30, 100

Chlortetracycline 100 10, 20, 30, 100 Table 2. Antibiotics tested with the Maximum Residue Limit (MRL) and the concentrations tested for each.

MATERIALS AND METHODS

Antibiotic stock solutions were prepared at 100-250 mg/L using USP grade antibiotic stocks when available (desfuroylceftiofur, and desacetylcephapirin were obtained from Toronto Research Chemical). Beta-lactam stock solutions were prepared in 50:50 (% v/v) methanol/water. Tetracyclines were prepared in 100% methanol (ACS grade). All solutions were mixed and sonicated until fully dissolved.

All tests were performed using a single milk sample (Lot # 91017OP) stored at - 80°C in single use aliquots. Each aliquot was thawed before use and then fortified with the appropriate concentration of the antibiotic stock solution.

Positive and negative samples were determined using the Qiagen ESE Reader Plus. A result was determined positive when the antibiotic to control line ratio was < 1.00, and determined negative when the antibiotic to control line ratio was ≥ 1.00.

RESULTS

For each antibiotic in the study, a dose response curve was generated at various concentrations at or around the MRL, using the intensity of the line to determine the limit of detection. Figures 2 through 21 illustrate this response using each class of antibiotics’ respective line intensity versus the control line.

Figure 2. Dose response curve for Amoxicillin versus Control.

Figure 3. Dose response curve for Ampicillin versus Control.

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Figure 4. Dose response curve for Cefacetrile versus Control.

Figure 5. Dose response curve for Cefalonium versus Control.

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Figure 6. Dose response curve for Cefazolin versus Control.

Figure 7. Dose response curve for Cefoperazone versus Control.

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Figure 8. Dose response curve for Cefquinome versus Control.

Figure 9. Dose response curve for Ceftiofur versus Control.

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Figure 10. Dose response curve for Cefalexin versus Control.

Figure 11. Dose response curve for Cephapirin versus Control.

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Figure 12. Dose response curve for Cloxacillin versus Control.

Figure 13. Dose response curve for Dicloxacillin versus Control.

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Figure 14. Dose response curve for Nafcillin versus Control.

Figure 15. Dose response curve for Oxacillin versus Control.

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Figure 16. Dose response curve for Desfuroylceftiofur versus Control.

Figure 17. Dose response curve for Penicillin G versus Control.

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Figure 18. Dose response curve for Desacetylcephapirin versus Control.

Figure 19. Dose response curve for Tetracycline versus Control.

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Figure 20. Dose response curve for Oxytetracycline versus Control.

Figure 21. Dose response curve for Chlortetracycline versus Control.

The limit of detection for each antibiotic was calculated as the lowest detectable concentration where there is a 95% chance of obtaining a positive result, summarized in Table 3. An additional 115 negative milk samples were tested to ensure the reproducibility as summarized in Table 4.

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Antibiotic EU MRL (µg/kg)

Detection Limit (LOD)

Amoxicillin 4 3.5-4

Ampicillin 4 3

Cefacetrile 125 20

Cefalonium 20 8

Cefazolin 50 40

Cefoperazone 50 2

Cefquinome 20 12-15

Ceftiofur 100 20

Cefalexin 60 1100

Cephapirin 60 5-6

Cloxacillin 30 3

Dicloxacillin 30 2

Nafcillin 30 5-6

Oxacillin 30 3

Desfuroylceftiofur 100 30

Penicillin G 4 2

Desacetylcephapirin 60 18-20

Tetracycline 100 12-15

Oxytetracycline 100 30

Chlortetracycline 100 20-30

Table 3. Detection limit capability of the AuroFlow™ PR1ME™ BT Combo.

Line Intensity (mV)

Control Ceftiofur Beta-lactam Tetracycline

Average 217.5 449.3 449.8 428.8

SD 13.4 28.2 23.7 35.1

% CV 6.2 6.3 5.3 8.2

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Ratio

Ceftiofur Beta-lactam Tetracycline

Average 449.3 449.8 428.8

SD 28.2 23.7 35.1

% CV 6.3 5.3 8.2

Table 4. Average line intensity and ratio of testing 115 negative milk samples at each respective line on the AuroFlow™ PR1ME™ BT Combo test strip.

CONCLUSIONS

The AuroFlow™ PR1ME™ BT Combo MRL Assay is able to detect 14 beta-lactam antibiotics under the European MRL levels, as well as the metabolites of ceftiofur and cephaprin below MRL levels. The assay can detect all 3 regulated tetracycline antibiotics under the European MRL.

The validation results indicate that the AuroFlow™ PR1ME™ BT Combo MRL Assay is appropriate for use as a screening test for beta-lactams and tetracyclines in raw commingled cow’s milk.

PerkinElmer, Inc. 7050 Burleson Road Austin, TX 78748 USA P: (888) 208-2246 or (+1) 707-8993 www.perkinelmer.com

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