Optimization and Validation of a New Microbial Inhibition Test for the Detection of Antimicrobial Residues in Living Animals Intended for Human Consumption.

Even though antibiotics are necessary in livestock production, they can be harmful not only due to their toxicity, but also in view of their contribution to the emergence of antimicrobial resistance. Screening tests based on microbial growth inhibition appeared to be useful tools to prevent its entry into the food chain. They have nevertheless been traditionally carried out post mortem, leading to great economical loss and harm to the environment in case a positive sample is found. Hence, the objective was to evaluate the use of a screening test as an ante mortem alternative for the detection of antibiotic residues in meat: thus, Explorer®-Blood test was optimized and validated. After adapting the procedure for matrix preparation, the assay parameters were assessed from 344 antibiotic-free blood serum samples. Limits of Detection (LoDs) were defined by spiking blood serum with several of the most common antimicrobials used in veterinary practice. LoDs were similar to those obtained for meat and were at or below the maximum residue limits set by EU legislation for muscle. Analyses of in vivo injected samples, previously characterized by LC-MS/MS, demonstrated the method's accuracy and proved that Explorer®-Blood can be considered a suitable alternative to conventional post mortem screening methods.

Validation of the Eclipse Farm 4G & COMET for Detection of Antibiotics in Raw Bovine Milk: AOAC Performance Tested MethodSM 022101.

BACKGROUND: The Eclipse Farm 4G test coupled to the COMET device allows for automatic and easy screening of a broad range of antimicrobials in raw bovine milk. All results obtained with this system are available in real-time through a smartphone application (App) and in the Test4all cloud platform. OBJECTIVE: The objective of this study was to validate Eclipse Farm 4G & COMET for the screening of antimicrobials in raw bovine milk according to AOAC Performance Tested MethodSM procedures. METHOD: The test is based on the inhibition of microbial growth of Geobacillus stearothermophilus in the presence of antimicrobials. When sample tubes are incubated in the absence of antibiotics, spores germinate and cells grow, changing the medium color from purple to yellow (negative result). The presence of antimicrobials in the milk sample hinders bacterial growth, preventing the tubes from changing color from purple to yellow (positive result). The COMET device automatically integrates all assay steps (incubation, time control, and results interpretation) and communications between the device, smartphone, and cloud. RESULTS: LODs and detection capabilities were confirmed to be at or below the European Union (EU) maximum residue limit (MRL) for most of the evaluated molecules representing the main families of antimicrobials (penicillins, cephalosporins, tetracyclines, sulfonamides, macrolides, and aminoglycosides). False-positive rates and the effect of potentially interfering substances showed sufficient selectivity/specificity. The Eclipse Farm 4G & COMET system was shown to be robust, consistent, and stable during shelf life. CONCLUSIONS: The Eclipse Farm 4G & COMET system has been shown to be suitable for screening antibiotics in raw bovine milk. HIGHLIGHTS: The assay is an automatic and easy system for the detection of a broad range of antibiotics in raw bovine milk consistent with EU MRLs and can provide results any time and anywhere through a smartphone App and a cloud platform.

Development of a novel duplex lateral flow test for simultaneous detection of casein and ß-lactoglobulin in food.

Milk by-products such as whey and caseinate are widely used as ingredients or processing aids in food industry. However, since they could cause allergic reactions they are included in Allergen Control Plans. ß-Lactoglobulin is the major whey protein and caseins are main proteins in milk. Selection of a unique target to analyze the presence of milk in foods could be insufficient when the source of milk proteins is unknown. A new test based on lateral flow immunocromatography that combines the simultaneous and independent detection of both proteins (ß-lactoglobulin and casein) in one rapid test was developed. The assay was validated according to AOAC guidelines being able to detect ß-lactoglobulin (0.5 ppm), casein (2 ppm), whey and powder milk (1-5 ppm). No cross-reactivity was found with a panel of 38 food commodities. The method is a rapid and suitable tool to identify milk proteins in processed food, ingredients, and rinsing water.

Validation of a rapid lateral flow method for the detection of cows' milk in water buffalo, sheep or goat milk.

For many years, the adulteration of milk from sheep, goats or water buffalos with cows' milk has been a widespread practice due to the higher cost of milk from those other species. Because of this, great concern has been shown by many Protected Designation of Origin councils that have to assure the quality and genuineness of the cheese produced by their associates. Therefore, the whole production chain needs analytical tools that allow the control of potential adulteration. Rapid methods to be used in the field are scarce and have not been validated according to international guidelines. The aim of this work has been to validate a rapid test based on lateral flow immunochromatography to detect cows' milk in milk from other species, including buffalo's milk, according to AOAC guidelines. No false-positive result was found after analysing 146 known negative samples from individual animals. The lowest level of adulteration with a Probability of Detection (POD) of 1.00 (confidence interval between 0.94 and 1.00) was found at 0.5% of cows' milk. This level is below the current EU allowed level of cows' milk, set at 1%. Variations in the time of assay, volume of the analysis buffer and different batches of the test were evaluated to detect any effect on the false-positive rate or on the limit of detection of the test. The effects of compositional factors (such as high level of fat, protein and somatic cell counts) were also evaluated. The new rapid test to detect cows' milk in milk from other species is shown to be an adequate tool to control milk quality in routine analysis. This kind of test is very easy to use and it can be performed by untrained staff during milk collection at the farm or upon arrival at dairies.

Validation of the Explorer® 2.0 test coupled to e-Reader® for the screening of antimicrobials in muscle from different animal species.

The Explorer(®) 2.0 tube test is a microbial inhibition test for the screening of antimicrobial residues in food samples. The new e-Reader(®) device coupled to Explorer(®) 2.0 operates by incubation at a selected temperature, determination of the endpoint of the assay and interpretation to generate results. This system was validated for muscle samples according to the European Commission Decision 2002/657/EC. Sensitivity towards 25 substances from several groups of antimicrobials was investigated in a first step. Detection capabilities for six substances representing the six major antimicrobial groups were also determined in bovine muscle. The detection capabilities for amoxicillin (10 µg l(-1)), cefalexin (200 µg l(-1)), doxycyclin (100 µg l(-1)), sulfamethazine (100 µg l(-1)), tylosin (100 µg l(-1)) and neomycin (200 µg l(-1)) were in all cases at or below the maximum residue limit (MRL). Specificity and applicability of the test were demonstrated with muscle samples from four animal species (bovine, porcine, ovine and poultry) and results were found to be satisfactory. Ruggedness was evaluated on negative and spiked samples with sulfamethazine as a representative antimicrobial. Neither false-positives nor false-negatives were detected when varying the sample volume, the time of pre-incubation, the temperature of incubation and the batch of the test. These results prove that Explorer(®) 2.0 coupled to e-Reader(®) is a valuable tool for the screening of a broad range of antimicrobials in muscle. This new methodology simplifies the analysis and increases the accuracy of interpretation of the test results since the endpoint of the assay is automatically determined and results are interpreted objectively.

Quantitative determination of the okadaic acid toxins group by a colorimetric phosphatase inhibition assay: interlaboratory study.

An interlaboratory collaborative study to validate a colorimetric phosphatase inhibition assay for quantitative determination of the okadaic acid (OA) toxins group in molluscs, OkaTest, was conducted. Eight test materials, including mussels, scallops, clams, and cockles, were analyzed as blind duplicates. Blank samples and materials containing different OA toxin levels ranging from 98 to 275 microg/kg OA equivalents were included. The study was carried out by a total of 16 laboratories from 11 different countries. Values obtained for repeatability relative standard deviations (RSDr) ranged from 5.4 to 11.2% (mean 7.5%). Reproducibility RSD (RSD(R)) values were between 7.6 and 13.2% (mean 9.9%). The Horwitz ratio (HorRat) values ranged between 0.4 and 0.6. A recovery assay was also carried out using a sample spiked with OA. A mean recovery of 98.0% and an RSD of 14.5% were obtained. The results obtained in this validation study indicate that the colorimetric phosphatase inhibition assay, OkaTest, is suitable for quantitative determination of the OA toxins group. OkaTest could be used as a test that is complementary to the reference method for monitoring the OA toxins group.

Single laboratory validation of a ready-to-use phosphatase inhibition assay for detection of okadaic acid toxins.

A phosphatase inhibition assay for detection of okadaic acid (OA) toxins in shellfish, OkaTest, was single laboratory validated according to international recognized guidelines (AOAC, EURACHEM). Special emphasis was placed on the ruggedness of the method and stability of the components. All reagents were stable for more than 6 months and the method was highly robust under normal laboratory conditions. The limit of detection and quantification were 44 and 56 µg/kg, respectively; both below the European legal limit of 160 µg/kg. The repeatability was evaluated with 2 naturally contaminated samples. The relative standard deviation (RSD) calculated was 1.4% at a level of 276 µg/kg and 3.9% at 124 µg/kg. Intermediate precision was estimated by testing 10 different samples (mussel and scallop) on three different days and ranged between 2.4 and 9.5%. The IC(50) values of the phosphatase used in this assay were determined for OA (1.2 nM), DTX-1 (1.6 nM) and DTX-2 (1.2 nM). The accuracy of the method was estimated by recovery testing for OA (mussel, 78-101%; king scallop, 98-114%), DTX-1 (king scallop, 79-102%) and DTX-2 (king scallop, 93%). Finally, the method was qualitatively compared to the mouse bioassay and LC-MS/MS.