Validation of SNAPduo Beta-Tetra ST Test Kit

The SNAPduo ßeta-Tetra ST (IDEXX Laboratories Inc. Westbrook, Maine, USA) is a competitive receptor test for the rapid and simultaneous screening of residues of ß-lactams (penicillins and cefalosporins) and tetracyclines in raw milk. The test is an improvement of the SNAPduo ß-lactam-Tetra test, because it is run with an incubation at ambient temperature (15-30ºC), so an incubator is no longer needed. The time to result for the assay is approximately 7 minutes (an estimate 45 seconds of milk flow time and 6 minute of incubation at ambient temperature to allow for color development). In addition, compared to current SNAPduo ß-lactam-Tetra test, the SNAPduo ßeta-Tetra ST expands the detection capability of ß-lactams in milk: more ß-lactam antibiotic residues can be detected by SNAPduo ßeta-Tetra ST at the MRL levels.

This new test was validated at ILVO-T&V (Technology & Food Science Unit of the Institute for Agricultural and Fisheries Research of the Flemish Community) according to Commission Decision 2002/657/EC and to the CRL guidelines for the validation of screening methods for residues of veterinary medicines (Anonymous, 2010). The following analytical parameters were checked: test specificity, detection capability, and test robustness (impact of deviation of the test protocol, impact of the milk composition, and batch differences of reagents). Further the suitability of the SNAPduo ßeta-Tetra ST to screen different milk types (UHT milk, sterilized milk, reconstituted milk powder, thawed milk) or milk from animal species other than the cow (goat, ewe, and mare) 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 two national ring trials and an international proficiency study.

The test was very specific; no interaction was noticed by compounds of antibiotic families or chemotherapeutics other than ß-lactams except for the interference by clavulanic acid. All ß-lactams with a Maximum Residue Limit (MRL) in milk were at least in 95% of the replicates detected at their respective MRL except for cefalexin (ccß >7,500 µg/kg) and the 4-epimer of chlortetracycline (ccß=175 µg/kg). The detection capability for doxycline, a compound without MRL in milk, was 25 µg/kg. The detection capability of the SNAPduo ST for ß-lactam antibiotics is comparable to the detection capability of the SNAP Beta-Lactam ST (Reybroeck & Ooghe, 2011).

The repeatability of the reader and the test were very good. The test result remained reliable even when performing the SNAPduo ßeta-Tetra ST with the timing changes of different steps in the testing protocol including delay in activation of SNAPduo device, delay after dissolving the pellet in the milk and delay of reading. Also, the impact of milk parameters (somatic cells, total bacterial count, fat and protein content, pH) was tested as part of the robustness testing. No interference by these milk parameters was noticed except for milk with a high number of somatic cells (> 10⁶ ml-1) (both channels), milk with a high protein content (>4 %) (both channels) or milk of pH 6.0 (ß-lactam channel). No significant differences were noticed in testing different milk types (raw milk, UHT-milk, sterilized milk, reconstituted milk powder, or thawed milk) and other species animal milk (goats’, mares’, or ewes’ milk). Therefore, SNAPduo ßeta-Tetra ST offers the flexibility of testing all different milks. No significant differences in testing capability were found between two batches of reagents of SNAPduo ßeta-Tetra ST.

Out of the data of this validation study it can be concluded that the SNAPduo ßeta-Tetra ST is a very fast, simple, and reliable test for the control of residues of ß-lactam and tetracycline antibiotics in tanker milk at the entrance. The test could also be used at farm level in order to prevent tanker milk contamination. The test is also giving reliable rest results for different milk types and milk of animal species different from the cow (goat, ewe, mare).