premi test – fast & easy antibiotic...
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R-Biopharm AGAn der neuen Bergstraße 1764297 Darmstadt, [email protected]
R-Biopharm AG
Premi®Test – fast & easyantibiotic screening
Speaker: Gilbert Garrido
Date: 29.09.2011
Place: Almaty, Kasachstan
Premi ®Test 08.04.2011
diseases„growth
promoter“antibiotics
too short withdrawal time
antibiotic residues in animal products
Health risk for consumers
Surveillance programs NOEL -> ADI -> MRLs
How to test?
Introduction
Technology
Premi®Test
Detection Limits
Instructions
Business
Summary
Premi ®Test 29.09.2011
Permitted substances
LEGISLATION
MRLMaximum Residue Limit
NOELNo Observed Effect Level
ADIAcceptable Daily Intake
Prohibited substances
MRPLMinimum Residue Performance Limit
Prohibition
State of the art methods
Testsystems
LODLimit of Detection
Validation
Safety factor 1000x
Average consumption
Monitoring of compliance by NRKP and ERKP
Premi ®Test 08.04.2011
EC 37/2010 example: Acetylicsalicylic acid
No entryNot for use in animals from which milk or eggs are produced for human consumption.
Not applicable
No MRL required
All food producingspeciesexcept finfish
Therapeuticclassification
Other provisions 14(7) EC 470/2009
Target Tissue
MRLAnimalspecies
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MRL = Maximum Residue Limit
Premi ®Test 08.04.2011
EC 37/2010 example: Ampicillin
Milk 4 µg/kg
Kidney50 µg/kg
Liver50 µg/kg
Fat50 µg/kg
Anti-infectiousagents/Antibiotics
For fin fish the muscle MRL relatesto ‘muscle and skin in natural proportions’.MRLs for fat, liver and kidney donot apply to fin fish. For porcine and poultry species thefat MRL relates to ‘skin and fat in natural proportions’. Not for use in animals from which eggs are produced for humanconsumption.
Muscle50 µg/kgAll food producingspecies
Therapeuticclassification
Other provisions 14(7) EC 470/ 2009
Target Tissue
MRLAnimalspecies
Introduction
Technology
Premi®Test
Detection Limits
Instructions
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MRL = Maximum Residue Limit
Premi ®Test 08.04.2011
EC 37/2010 Prohibited substances
Pharmacologically active substance MRL
Aristolochia spp. and preparations thereof MRL can not be establishedChloramphenicol -Chloroform -Chlorpromazine -Colchicine -Dapsone -Dimetridazole -Metronidazole -Nitrofurans (including furazolidone) -Ronidazole -
Introduction
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Premi®Test
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-> MRPL etablished
Premi ®Test 08.04.2011
Introduction
Technology
Premi®Test
Detection Limits
Instructions
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Summary
Premi ®Test 08.04.2011
Premi ®Test technology
H+
H+
H+
H+
H+
Bacillus stearothermophilus
pH-Indicator Bromocresol
NEGATIVE POSITIVE
antibiotic residues
Introduction
Technology
Premi®Test
Detection Limits
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Summary
Premi ®Test 08.04.2011
Premi ®Test consists of:
Polystyrene package25 Test-AmpoulesPerforated foil1 Syringe30 TippsManual in 6 languagesColour chart
Introduction
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Premi ®Test 08.04.2011
µg / kg =ppb
RBP 31/02 – 04/11 ALTERNATIVE ANALYTICAL METHODS FOR AGRIBUSINESS Certified by AFNOR Certificationwww.afnor-validation.com
beef / pork / chickenβ-lactamsMacrolidesTetracyclinsSulphonamides
beef:Penicillin
Introduction
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Premi ®Test 08.04.2011
Introduction
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Premi®Test
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Instructions
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µg / kg =ppb
Premi ®Test 08.04.2011
Matrices:• Meat (beef, pork, poultry)• Fish• Shrimps• Kidney & Liver• Egg• Feed (pork/poultry)• Urine (beef/pork)• Plasma/Serum
Workflow
Sample Preparation
Colour Reading
Incubation
Introduction
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Premi ®Test 08.04.2011
What you need for Premi ®Test:
Starter Kit• Briefcase• Incubator• Meatpress• Scissors• Lab-Timer
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Premi ®Test 08.04.2011
Sample preparation
Cut off the required number of ampoules (number of samples + negative control)
Essential:Negative control = sample of the same matrix/species which contains no antibiotics
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Premi ®Test 08.04.2011
Sample preparation
Take approx. 2 cm 3 of meat and use the meatpress to extract meatjuice.
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Premi ®Test 08.04.2011
Sample preparation
In case you have to analyze a large number of samples, the Multipress allowsto squeeze 12 samples at once.
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Premi ®Test 08.04.2011
Instructions for use
Pipette 100 µl of thesample slowly onto the agar in the ampoule.
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Premi ®Test 08.04.2011
Instructions for use
Allow to stand at room temperature for 20 minutes for pre diffusion of the antibiotic residues into the agar.
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Premi ®Test 08.04.2011
Instructions for use
Flush the sample away by gently washing the test twice with demineralised water and remove the water from the test carefully.
Introduction
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Premi ®Test 08.04.2011
Instructions for use
Close the test ampoule with the supplied foil to avoid evaporation and start incubation.
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Premi ®Test 08.04.2011
1. Squeeze the sample with the meatpressor the multipress to obtain juice
2. Freeze and thaw the sample and collectthe released juice
3. Incubate the samples in a ziplock bag in a waterbath at 64°C for 10 / 5 min and collect the released juice (preservatives?)
1. Homogenize whole egg
Sample preparation: Meat, fish, shrimps
Sample preparation: Egg
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Premi ®Test 08.04.2011
1. Cut the kidney horizontally.2. Cut a piece of around 1-2 cm3 out
of the medulla.3. Squeeze the kidney sample with
the meatpress or Multipress to obtain ‚kidney‘ juice
1. Grind the liver to obtain a paste2. Dilute the liver-paste 1:1 with
demineralised water3. Mix well to a homogeneous
mixture
Sample preparation: Kidney
Sample preparation: Liver
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Premi ®Test 08.04.2011
1. Grind the feed.2. Mix 10 g of the grinded feed with
30 ml of demineralized water3. Stir for half an hour at room
temperatureproblems with complex matrices (rice bran)
protocol in validation process
Sample preparation: poultry/pig feed
Sample preparation: Honey
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Premi ®Test 08.04.2011
Sample preparation – Urine, Plasma
Premi ®Test Urine20 ampoules for thepretreatment of cattle/pork urineor serum/plasma
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Premi ®Test 08.04.2011
workflow
Sample Preparation
Colour Reading
Incubation
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Premi ®Test 08.04.2011
Incubation
2-Step Incubationshrimps, egg, feed, kidney
10 min at 80 °Capprox. 3 h at 64°C
1-Step Incubationmeat, fish, liver, urine, serum
approx. 3 h at 64 °C
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Premi ®Test 08.04.2011
Workflow
Sample Preparation
Colour Reading
Incubation
Introduction
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Premi ®Test 08.04.2011
Withdraw the sample ampoules from the incubator when the negative control changed its colour into yellow(approx. 3 h).
Instructions for useIntroduction
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Premi ®Test 08.04.2011
Determination of results: by eye
positiveconc. > LOD
negativeconc. < LOD
pos/negconc. = LOD
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Premi ®Test 08.04.2011
Determination of results: Premi ®Scan
Premi ®Scan• Premi ®Scan Software• 2 templates• Kodak colour chart for
calibration• Black Cloth• not supplied: Scanner
Software for interpretation & documention of results
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Premi ®Test 08.04.2011
Determination of results: Premi ®Scan
Position Description z-value ResultA1 Sample 1 -12,35 NEGB1 Sample 2 11,84 POS
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Premi ®Test 29.09.2011
Title Authors(s) JournalScreening methods for the detection of antibiotic residues in slaughter animals: comparison of the European Union Four-Plate Test, the Nouws Antibiotic Test and the Premi®Test (applied to muscle and kidney).
Pikkemaat MG, Rapallini ML, Zuidema T, Elferink JW, Oostra-van Dijk S, Driessen-van Lankveld WD.
Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2011 Jan;28(1):26-34.
Microbial screening methods for detection of antibiotic residues in slaughter animals. Pikkemaat MG. Anal Bioanal Chem. 2009 Oct;395(4):893-905. Epub 2009 May 30.
Evaluation of three different microbial inhibition tests for the detection of sulphamethazine residues in the edible tissues of rabbit.
Kozárová I, Janosová J, Máté D, Tkáciková S. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2009 Jul;26(7):978-87.
Comparative study of three screening tests, two microbiological tube tests, and a multi-sulphonamide ELISA kit for the detection of antimicrobial and sulphonamide residues in eggs.
Gaudin V, Hedou C, Rault A, Sanders P, Verdon E. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2009 Apr;26(4):427-40.
Comparison of screening methods for antibiotics in beef kidney juice and serum. Schneider MJ, Mastovska K, Lehotay SJ, Lightfield AR, Kinsella B, Shultz CE.
Anal Chim Acta. 2009 Apr 1;637(1-2):290-7. Epub 2008 Aug 19.
AFNOR validation of Premi Test, a microbiological-based screening tube-test for the detection of antimicrobial residues in animal muscle tissue.
Gaudin V, Juhel-Gaugain M, Morétain JP, Sanders P. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2008 Dec;25(12):1451-64.
New method for the rapid identification of tetracycline residues in foods of animal origin - using the PremiTest in combination with a metal ion chelation assay.
Stead SL, Caldow M, Sharma A, Ashwin HM, Sharman M, de-Rijk A, Stark J.
Food Addit Contam. 2007 Jun;24(6):583-9.
Evaluation of the EEC four-plate test and Premi test for screening antibiotic residues in trout (Salmo trutta)
Berna Kilinc1,*,Carsten Meyer2,Volker Hilge Int J Food Sci Technol (2007) 42:635-628
Improved microbial screening assay for the detection of quinolone residues in poultry and eggs.
Pikkemaat MG, Mulder PP, Elferink JW, de Cocq A, Nielen MW, van Egmond HJ
Food Addit Contam. 2007 Aug;24(8):842-50.
Comparison of various assays used for detection of beta-lactam antibiotics in poultry meat.
Popelka P, Nagy J, Germuska R, Marcincák S, Jevinová P, De Rijk A.
Food Addit Contam. 2005 Jun;22(6):557-62.
Use of Premi®Test for the detection of sulphonamide residues in chicken eggs Hussein K.a, Marcinc(ák S.a, Máté D.a, Kožárová Ivonaa, Sokol J.a, Zdolec N.b
Acta veterinaria 2005, vol. 55, iss. 5-6, pp. 493-500
Meeting maximum residue limits: an improved screening technique for the rapid detection of antimicrobial residues in animal food products.
Stead S, Sharman M, Tarbin JA, Gibson E, Richmond S, Stark J, Geijp E.
Food Addit Contam. 2004 Mar;21(3):216-21.
Evaluation and establishing the performance of different screening tests for tetracycline residues in animal tissues.
Okerman L, Croubels S, Cherlet M, De Wasch K, De Backer P, Van Hoof J.
Food Addit Contam. 2004 Feb;21(2):145-53.
Premi ®Test publications
Premi ®Test 08.04.2011
R-Biopharm product portfolio for antibiotic testing
Premi®Test:broad spectrum antibiotic screening test
RIDASCREEN® :ELISAs for specific antibiotic screening
EASY EXTRACT® Immunoaffinity columnsfor use in conjunction with an HPLC or LC-MS/MS system
Introduction
Technology
Premi®Test
Detection Limits
Instructions
Business
Summary
Premi ®Test 08.04.2011
Production
Distribution
Premi ®Testantibiotic
screening forall matricesexcluding
dairy
Delvo ®Testantibiotic
screening fordairy
products
Premi ®TestSalmonella
Salmonellaserotyping
Situation until 31st Dec 2010Situation since 1st Jan 2011Introduction
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Premi®Test
Detection Limits
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Premi ®Test 08.04.2011
Premi ®Test products
Premi ®Test 25R3925
Premi ®Test 4x25R3900
Premi ®Test UrineR3920
Starter KitZPT2000
MultipressZPT2012
Premi ®ScanZPT2010
Introduction
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Premi®Test
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Premi ®Test 08.04.2011
SUMMARY
Premi ®Test is
• FAST < 4 hours (test implementation: ½ h + incubation: 3 h)
• EASY simple handling and no cost intensive equipment nee ded
• SENSITIVE detects antibiotic residues in line with MRL’s
• RELIABLEDSM - 28/1 - 06/06ALTERNATIVE ANALYTICAL METHODS FOR AGRIBUSINESS Certified by AFNOR Certificationwww.afnor-validation.comFin de validité: 30.08.2014
Introduction
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Premi ®Test 08.04.2011