validation of the twinsensor milk for rapid screening...

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VALIDATION OF THE TWINSENSOR MILK FOR RAPID SCREENING OF ββββ-LACTAMS

AND TETRACYCLINES IN MILK

Wim Reybroeck and Sigrid OogheT&V-ILVO Melle

5th International Symposium on Hormone and Veterinary Drug Residue Analysis

Antwerp, Belgium 19/05/2006


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TESTING UPON THE DAIRY COMPANY(self control)

CONTROL ON RECEPTION OF THE MILK OR BEFORE PRODUCTION

⇒ protection of the fermentation process

⇒ EEC-Directive 92/46

• self control (own checks)

• >MRL: milk not suitable for human

consumption (destruction)


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WHICH RESIDUES DO WE NEED TO DETECT?• Study of milk samples resulting in penalisation based on a positive

Delvotest MCS result (May & June 2003, Flanders) (Reybroeck and Daeseleire, 2003)

1° penicillin G 2° cloxacillinIn some countries (e.g. France) tetracyclines are also often found in milk

IDENTIFICATION OF INHIBITORS9%

4%

8%

79%

β-lactam, non synthetic penicillins

β-lactam, synthetic penicillins and cefalosporins

combination β-lactam/non β-lactam

non β-lactam (sulfonamides,…)


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TWINSENSOR MILK (Unisensor s.a., Belgium)

Competitive receptor assay in dipstick format for the simultaneous detection of β-lactams and tetracyclines in milk in 6 minutes.


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TWINSENSOR MILK: KIT VALIDATION ACCOR-DING TO COMMISSION DECISION 2002/657/EC

- Specificity / cross-reactivity- Detection capability- Test ruggedness

- Test performance influences- Length of incubation- Influence of waiting time on reader results

- Reagents (under evaluation)- Batch differences- Test kit stability

- Milk parameters- Impact of somatic cells, bacterial count, fat content, protein content, pH- Type of milk (raw, pasteurised, UHT, sterilised)- Milk from animal species other than the cow (ewes, goats, mares)

- Test for false positive / false negative results- International ring trial


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TWINSENSOR MILK: TEST PRINCIPLECompetitive receptor test in dipstick format

1st step: microplate cup with receptor + 200 µl of milk (3 min at 50°C)

β-lactam antibiotics + receptor ⇒ stable non-active complextetracyclines + receptor ⇒ stable non-active complex

2nd step: addition of a dipstick, migration and colour formation(3 min at 50°C)

→ ‘CTRL’ line captures a part of the excess of reagents

→ ‘TEST’ line captures remaining active receptor for β-lactams

‘TEST’ line captures remaining active receptor for tetracyclines→


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TWINSENSOR MILK: VISUAL READING

TEST Tetra→CTRL →TEST β-L →

POS

POS

NEGPOSNEGunvalidResultββββ-Lactams

POSNEGNEGunvalidResult Tetracyclines


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TWINSENSOR MILK: INSTRUMENTAL READINGREAD SENSOR: result = ratio test line / control line+ RAPID software- area or amplitude

> 1.10: ‘neg’> 0.90 & ≤ 1.10: ‘low pos’≤ 0.90: ‘pos’


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TEST KIT SPECIFICITY / CROSS-REACTIVITY

Small interference by clavulanic acid at 2000 µg/kg on β -lactam channel.

⇒ The TWINSENSOR MILK assay is very specific for the analysis of β -lactam / tetracyclines in milk.

trimethoprimdiamino pyrimidine derivates

clavulanic acidβ-lactamase inhibitors

sulfadiazinesulphonamidesenrofloxacinquinolones

neomycinaminoglycosideserythromycinmacrolides

lincomycinlincosamidesoxytetracyclinetetracyclines

chloramphenicolamphenicolscefaloniumcephalosporins

colistinpolymyxinspenicillin Gpenicillins

substancegroup Substancegroup

Milk spiked at 10x MRL (or 10x MRPL) with one compound of the most important groups of antibiotics or chemotherapeutics


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TEST KIT SENSITIVITY• All penicillins, cefalosporins and tetracyclines on the MRL list

tested• Spiked in raw blank milk

– range 1 -10 ppb: in steps of 1 ppb– range 10 - 20 ppb: in step of 2 ppb– range 20 - 50 ppb: in step of 5 ppb– range 50 -100 ppb: in steps of 10 ppb

• 20 Replicates• Blind coded, randomly mixed

• Test kit sensitivity defined as the lowest concentration tested giving at least 19 upon 20 times a positive result.


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TWINSENSOR MILK: TEST SENSITIVITYMRLs and test sensitivities in µg/kg

18

10

6

50

6

6

14

4

3

2

Test Sensitivity

> 200100cefalexine4ampicillin

35125cefacetrile4amoxicillin

14---doxycycline50cefazolin

25100chlortetracycline100ceftiofur

30100oxytetracycline60cephapirin

40100tetracycline30nafcillin

30dicloxacillin

420cefalonium30cloxacillin

350cefoperazone30oxacillin

3020cefquinome4penicillin G

Test Sensitivity

MRLSubstanceMRLSubstance

⇒ The TWINSENSOR MILK detects all β-lactam (excepted for

nafcillin, cefquinome and cefalexine) and tetracyclines in milk at MRL.


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TWINSENSOR MILK : TEST SENSITIVITYIn between brackets: MRLs in µg/kg.

Inner circle = 2 MRL, circle 2 = MRL, circle 3 = 0.5 MRL, outer circle = 0.25 MRL

Beta-lactam compoundspenicillin G (4)

ampicillin (4)

amoxicillin (4)

oxacillin (30)

cloxacillin (30)

dicloxacillin (30)

nafcillin (30)

cephapirin (60)ceftiofur (100)

cefazolin (50)

cefquinome (20)

cefalexin (100)

cefacetrile (125)

cefoperazone (50)

cefalonium (20)


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TEST RUGGEDNESS – TEST PARAMETERSINFLUENCE LENGTH OF INCUBATION PERIODS• Reading T1/C & T2/C after different incubation periods• Blank and doped farm milk (3 ppb pen G & 30 ppb OTC)

1.631.851.400.710.670.574 doped

1.081.291.200.410.630.493 doped

1.211.481.450.680.800.721 doped

1.431.631.610.530.610.552 doped

2.442.571.932.412.522.064

2.012.602.332.202.321.973

1.992.482.322.252.472.262

1.942.202.112.202.442.371

3’ + 2’302’30 + 3’3’ + 3’3’ + 2’302’30 + 3’3’ + 3’

tetracyclinesβ-lactamfarm milk


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1.911.531.400.550.630.574 doped

1.401.291.200.470.450.493 doped

1.701.441.450.550.540.721 doped

1.861.671.610.420.440.552 doped

3.092.521.932.792.582.064

2.762.392.332.192.231.973

2.722.652.322.502.632.262

2.702.232.112.732.382.371

3’ + 3’303’30 + 3’3’ + 3’3’ + 3’303’30 + 3’3’ + 3’



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2.041.891.400.570.640.574 doped

1.581.401.200.440.600.493 doped

1.661.531.450.530.600.721 doped

2.071.891.610.530.590.552 doped

2.922.821.932.692.872.064

2.882.622.332.262.511.973

2.812.682.322.772.652.262

2.442.392.112.662.452.371

4’ + 4’3’30 + 3’303’ + 3’4’ + 4’3’30 + 3’303’ + 3’



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TEST RUGGEDNESS – TEST PARAMETERSINFLUENCE OF WAITING TIME ON RESULTS• Reading T1/C & T2/C after 0, 100 and 200 seconds• Blank and doped farm milk (3 ppb pen G & 30 ppb OTC)

1.671.461.041.241.121,014 doped

1.841.591.250.770.750.713 doped

0.970.850.590.230.230.191 doped

1.601.361.010.660.650.592 doped

2.912.512.222.832.592.414

2.922.501.822.492.322.013

3.282.781.912.812.662.152

2.652.381.791.501.451.251

t=200’’t=100’’t=0t=200’’t=100’’t=0



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TEST RUGGEDNESS – MILK PARAMETERSIMPACT OF SOMATIC CELLS• 36 milk samples with SCC > 1.106 / ml7 samples positive for β-lactam (3 positive with βeta-s.t.a.r.)4 samples positive for β-lactam + tetracyclines (0 positive with βeta-s.t.a.r.)

LC-MS/MS analysis (Dr. E. Daeseleire) of 2 positive samples: no presence of β-lactam antibiotics

• 4 normal milk samples + 10 milk samples with high SCC,also doped with 3 ppb pen G & 40 ppb OTC

tetracyclinesβ-lactam

1.240.450.660.730.230.37doped in milk with high SCC

1.230.901.080.630.300.44doped in normal milk

2.951.902.432.951.271.91milk with high SCC (n=10)

2.582.212.362.212.052.14normal milk (n=4)

maxminavgmaxminavg

samples


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TEST RUGGEDNESS – MILK PARAMETERS

The impact of following milk parameters on the TwinSensor results isstill under investigation:

- bacterial count- fat content- protein content- pH- type of milk (pasteurised,UHT, sterilised, milk powder)- milk from animal species other than the cow (ewes,

goats, mares)


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TEST RUGGEDNESS – TEST FOR FALSE POSITIVE / FALSE NEGATIVE RESULTS

• TWINSENSOR MILK integrated in monitoring program (farm milk, truck milk)

• Other screening tests:– Delvotest SP-NT– Bacillus cereus-test (tetracyclines)– Escherichia coli- test (quinolones)

– βeta-s.t.a.r./ Charm MRL Beta-lactam Test (ROSA) (cefquinome)

• So far, no false negative test results, no false positivetest results.


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INTERNATIONAL RING TRIALLIST OF PARTICIPATING LABS (16)

- AFFSA, Community reference laboratory for antimicr obial residues, France- Faculty of Veterinary Medicine, University of Belgr ade, Serbia and Montenegro- Fed. Research Centre for Nutrition & Food, Inst. Hygi ene & Food Safety, Germany- Istituto Superiori di Sanità – CNQARA, Italy- Laboratorio Agroalimentario de Santander, Spain - National Food Administration, Chemistry Division 1 , Sweden- National Milk Laboratories, United Kingdom- Nat. Vet. Research Inst., Department of Hygiene of Food of Animal Origin, Poland- Nestlé Research Center; Switzerland- Scientific Institute of Public Health, Belgium- State Veterinary Institute Jihlava, Czech Republic- Univ. in Ljubljana, Biotech. Faculty, Zootechnical D ep., Lab. for Dairying, Slovenia- University of Veterinary Medicine, Department of V eterinary Public Health, Austria- Univ. Zagreb, Fac. of Food Tech. and Biotech., Resid ue Analysis Center, Croatia

- Unisensor s.a., Belgium

- T&V-ILVO, Melle, Belgium


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INTERNATIONAL RING TRIALCODIFICATION + RESULTS (visual reading)

results (number of labs)

ββββ -lactam tetracyc lines

code substance(s) conc.

(µg/kg)

MRL

(µg/kg)

neg pos neg pos

T1 = T18 c loxac illin 15 30 32x 32x

T2 = T11 blank m ilk --- --- 32x 31x 1x (lab 10)

T3 = T16 cefoperazone 5 50 1x (lab 10) 31x 31x 1x (lab 10)

T4 = T12 penic illin G 4 4 32x 32x

T5 = T20 te tracycline 75 100 32x 32x

T6 = T15 amoxic illin 6 4 32x 32x

T7 = T13 ceftio fur 30 100 32x 32x

T8 = T19 sulfamethazine 1000 --- 32x 32x

T 9 = T17 penic illin G

nafc illin

2

20

4

30

1x (lab 3) 31x 32x

T10 =T14 oxytetracyc line

penic illin G

50

3

100

4

1x (lab 10)

31x

1x (lab 10) 31x

In general very favourable results.4 false negative results (3x lab 10 and 1x lab 3); 2 false positive results (lab 10).


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(µg/kg)

MRL

(µg/kg)

neg pos neg pos







T7 = T13 ceftio fur 30 100 32x 32x



nafc illin

2

20

4

30

1x (lab 3) 31x 32x


penic illin G

50

3

100

4

1x (lab 10)

31x

1x (lab 10) 31x


Samplewith thelowest stability


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(µg/kg)

MRL

(µg/kg)

neg pos neg pos







T7 = T13 ceftio fur 30 100 32x 32x



nafc illin

2

20

4

30

1x (lab 3) 31x 32x


penic illin G

50

3

100

4

1x (lab 10)

31x

1x (lab 10) 31x


Samplewith thelowest stability

Lack of training?Language problem?


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INTERNATIONAL RING TRIALRESULTS (instrumental reading) OF 3 LABS

ß-lactam TetraT&V-ILVO UNISENSOR NESTLE T&V-ILVO UNISENSOR NESTLE

RATIO RATIO RATIO RATIO RATIO RATIOBlank 2,46 2,84 3,70 2,10 2,12 3,55

PEN 4ppb 0,34 0,15 0,53 2,59 2,74 3,75T1 0,56 0,35 0,65 2,47 2,28 3,58T2 2,76 2,80 3,66 2,42 2,16 3,55T3 0,53 0,36 0,57 2,27 2,28 3,29T4 0,40 0,26 0,52 2,71 2,56 3,62T5 2,63 2,83 3,38 0,53 0,59 0,82T6 0,58 0,62 0,81 2,79 2,61 3,65T7 0,70 0,59 0,70 2,25 2,27 3,45T8 2,64 2,85 3,42 1,62 2,24 3,14T9 0,87 0,67 0,67 1,89 2,41 2,94T10 0,71 0,31 0,52 0,56 0,66 0,94T11 2,58 2,73 3,43 1,44 1,91 3,43T12 0,49 0,15 0,46 2,06 2,40 3,45T13 0,83 0,56 0,79 1,91 2,29 3,59T14 0,63 0,20 0,52 0,54 0,69 0,89T15 0,75 0,59 0,70 1,77 2,43 3,31T16 0,51 0,19 0,57 2,43 2,51 3,48T17 0,82 0,70 0,65 2,25 2,44 3,18T18 0,62 0,41 0,62 2,62 2,49 3,31T19 2,63 2,97 3,59 2,03 2,26 3,35T20 2,91 2,87 3,41 0,62 0,63 0,93


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CONCLUSION

The TWINSENSOR MILK is a suitable test for rapid screening of milk on the presence ofβ-lactam + tetracyclines

Some ruggedness parameters are still under investigation- Reagents

- Batch differences- Test kit stability

- Milk parameters- Impact of bacterial count, fat content, protein content, pH- Type of milk (raw, pasteurised, UHT, sterilised)- Milk from animal species other than the cow (ewes, goats, mares)


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Thank you foryour attention

β -lactam tetra


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All rights reserved Tel: +32 9 272 30 11© Wim Reybroeck Fax: +32 9 272 30 01T&V-ILVO 2006 E-mail: [email protected]

Brusselsesteenweg 370 B-9090 MELLE, Belgium

Oral presentation given on May 19th 2006 at the 5th International Symposium on Hormone and Veterinary Drug Residue Analysis in Antwerp, Belgium

validation of the twinsensor milk for rapid screening...

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