Validation of the AnticFast® Beta-Lactams & Tetracyclines Combo Test Kit for Detection of Residues of Beta-Lactams (Penicillins and Cefalosporins) and Tetracyclines in Raw Cows' Milk: AOAC Performance Tested MethodSM 032403.

BACKGROUND: The AnticFast® Beta-lactams & Tetracyclines Combo Test Kit is a qualitative two-step (2 min + 5 min) rapid lateral flow assay to detect ß-lactam (penicillins and cefalosporins) and tetracycline antibiotic residues in raw commingled cows' milk. OBJECTIVE: The method performance was evaluated according to Commission Implementing Regulation 2021/808 and Community Reference Laboratories Residues Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines and submitted for AOAC Performance Tested MethodsSM certification. METHODS: The AnticFast Beta-lactams & Tetracyclines Combo Test Kit was evaluated for detection capability (CCß), selectivity, false-positive results, repeatability, robustness, suitability for various milk types and milk compositions, milks from various species, and test kit consistency and stability. Samples included milks spiked at concentrations bracketing the EU maximum residue limits (MRLs) for ß-lactams and tetracyclines as well as bulk farm and tanker milks. RESULTS: The AnticFast Beta-lactams & Tetracyclines Combo Test Kit is specific for the detection of residues of ß-lactams and tetracyclines in milk and does not detect residues from other antibiotic families. Interference was seen with clavulanic acid, a ß-lactamase inhibitor, which was expected. The test can detect (minimum a 95% detection) all residues of ß-lactams (penicillins and cepfalosporins) and tetracyclines (parent drugs and their 4-epimers) present on the EU-MRL list for milk at their respective MRL except for desfuroylceftiofur and cefalexin, with a 95% detection only above the MRL. No false positives were detected in 599 blank samples (out of 305 farm and 301 tanker load samples) tested on both channels. Five real positives were detected and confirmed on the tetracycline channel for the farm milk samples, and two positives were detected and confirmed on the ß-lactam channel for the tanker samples. Robustness testing indicated that the detection in high-protein raw cows' milk and heat-treated milk types (UHT, sterilized, and reconstituted milk powder) may be slightly hampered. For substances with a detection capability well below the MRL, this interference does not cause problems since detection at MRL remains guaranteed, but care should be taken for substances with a CCß at or near their MRL. Diminished sample flow was seen for high-fat raw cows' milk and for all other cows' milk types other than raw milk and blank ewes' milk, so sample flow should always be verified for these milk types. CONCLUSIONS: Results of this validation show that the AnticFast Beta-lactams & Tetracyclines Combo Test Kit is a reliable test for rapid screening of raw cows' milk for residues of ß-lactam and tetracycline antibiotics. HIGHLIGHTS: The AnticFast Beta-lactams & Tetracyclines Combo Test Kit is an easy, reliable, robust, and highly specific test for screening of ß-lactam (penicillins and cefalosporins) and tetracycline antibiotic residues in milk with incubation at room temperature. In raw cows' milk, all tetracyclines are detected below 10 µg/kg.

Validation of the AnticFast® Beta-Lactams Rapid Test Kit for Detection of Beta-Lactams (Penicillins and Cephalosporins) in Raw Cow's Milk: AOAC Performance Tested MethodSM 032303.

BACKGROUND: AnticFast® Beta-Lactams Rapid Test Kit is a qualitative two-step (2 min + 5 min) rapid lateral flow assay to detect ß-lactam (penicillins and cephalosporins) antibiotic residues in raw commingled cow's milk. OBJECTIVE: The method performance was evaluated according to Commission Decision 2002/657/EC, Commission Implementing Regulation 2021/808, and Community Reference Laboratories Residues Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines. METHODS: The AnticFast Beta-Lactams Rapid Test Kit was evaluated for detection capability, selectivity, false-positive results, repeatability, robustness, suitability for various milk types and milk compositions, milks from various species, and test kit consistency and stability. Samples included milks spiked at concentrations bracketing the EU maximum residue limits (MRLs) for ß-lactams as well as bulk farm and tanker milks. RESULTS: The AnticFast Beta-Lactams Rapid Test Kit is specific for the detection of ß-lactams in milk and does not detect compounds from other antibiotic families. Interference was seen with clavulanic acid, a ß-lactamase inhibitor, which was expected. The test can detect all residues of ß-lactams (penicillins and cephalosporins) present on the EU-MRL list for milk at their respective MRL except for desfuroylceftiofur and cephalexin, which were above the MRL. No false positives were detected in the 602 (300 blank farm and 302 tanker load) samples tested. Robustness testing indicated that the detection in heat-treated milk types may be slightly hampered. For substances with a detection capability well below the MRL, this interference does not cause problems since detection at MRL remains guaranteed, but care should be taken for substances with a CCß at or near their MRL. Diminished sample flow was seen with reconstituted milk powder and blank ewes' milk, so sample flow should always be verified for these milk types. CONCLUSIONS: Results of this validation show that the AnticFast Beta-Lactams Rapid Test Kit is a reliable test for rapid screening of raw cows' milk for residues of ß-lactam antibiotics. HIGHLIGHTS: AnticFast Beta-Lactams Rapid Test Kit is an easy, realiable, robust and highly specific test for screening of raw cows' milk for residues of penicillins and cephalosporins.

Validation of a lateral flow test (MRLAFMQ) for the detection of aflatoxin M1 at 50 ng l⁻¹ in raw commingled milk.

Aflatoxin M1 contamination in dairy products is a risk when feedstuff contaminated with aflatoxin B1 produced by moulds is consumed by milk-producing animals. Milk can be screened for aflatoxin M1 at the European Union maximum limit of 50 ng l⁻¹ by a lateral flow test, the MRLAFMQ (Aflatoxin M1) Test. The method takes 15 min with no milk dilution or a sample preparation step. The lateral flow assay was validated at the Technology and Food Science Unit of the Institute for Agricultural and Fisheries Research (ILVO-T&V) according to European Union guidelines using fortified raw milk samples. A detection capability of 50 ng l⁻¹ was demonstrated with a false negative rate lower than 2% at 50 ng l⁻¹ and a false positive rate of less than 0.3%. Quantitative readings had a mean bias of +2 to 6 ng l⁻¹ at 50 ng l⁻¹ with a standard deviation of 5-8 ng l⁻¹. Based on the validation results, the test could be considered appropriate for milk screening prior to milk unload at dairies.

Validation of the Charm MRL-3 for fast screening of beta-lactam antibiotics in raw milk.

The biochemicals utilized in the Charm MRL beta-Lactam test (8 min test) were applied to faster flowing lateral components to create a new 3 min, one-step beta-lactam test called Charm MRL-3 (Charm Sciences Inc., Lawrence, MA). This new test was validated at T&V-ILVO according to Commission Decision 2002/657/EC. The following analytical parameters were checked: test specificity, detection capability, and test robustness (impact of deviation of the test protocol, and impact of the milk composition, batch differences of reagents). Further, the suitability of the Charm MRL-3 to screen heat-treated milk or milk from animal species other than the cow was also tested. Finally, the test was integrated in the monitoring of dairy samples to check the occurrence of false-negative or false-positive results, and the test was also included in a national ring trial and an international proficiency study. The results proved that the Charm MRL-3 is a fast, simple, and reliable cows' milk test that can be used at the farm level in order to prevent tanker milk contamination, or at the entrance of the dairy plant to screen tanker milk for the presence of beta-lactam antibiotics.

Validation of the tetrasensor honey test kit for the screening of tetracyclines in honey.

Regarding anti-infectious agents, no maximum residue limits are fixed for honey in the European legislation. Discussions are being conducted in order to set working limits at the European level; for example, for tetracyclines, 20 microg/kg was proposed. The Tetrasensor Honey test kit is a receptor-based assay using dipsticks for a rapid screening (30 min) of honey on the presence of tetracyclines. The test was validated according to Commission Decision 2002/657/EC. The test detects tetracycline, oxytetracycline, chlortetracycline, and doxycycline in honey in a specific and sensitive way. Depending on the type of tetracycline, detection capabilities (CCbeta) between 6 and 12 microg/kg were obtained (4-7 microg/kg for dried dipsticks). The test is rugged and participation with the test in an international ring trial gave compliant results. It can be concluded that the Tetrasensor Honey test kit is a simple and reliable test that can even be used at the production site.