Novel bioassay using Bacillus megaterium to detect tetracycline in milk (original) (raw)
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Testing of Raw Milk for Tetracycline Residues
Journal of Dairy Science, 1998
A newly improved Bacillus calidolactis tube diffusion test and two postscreening test systems-a receptor assay (Charm HVS; Charm Sciences, Inc., Malden, MA) and a newly developed Bacillus cereus ATCC 11778 mycoides test system-were evaluated for the detection and identification of tetracycline residues using 973 samples of bulk milk taken at random in The Netherlands. All milk samples were assayed with the B. calidolactis tube and the receptor test. The milk samples testing as suspect or positive with one or both of the test systems were analyzed with HPLC (limit of detection, 10 ng/ml) and the recently developed B. cereus test system. The B. calidolactis tube diffusion test detected tetracycline residues >45 ng/ml in milk. With the B. cereus test plate, residues of oxytetracycline and tetracycline of >30 ng/ml milk were detected; for chlortetracycline and doxycycline, the detection limit was 10 ng/ml. Raw milk exhibiting inhibition diameters of <20 mm on the B. cereus test plate fulfilled the European Union criterion for maximum residue level for tetracyclines of <100 ng/ml (including their 4-epimer derivatives). The detection limits of the receptor assay depended on the type of milk used. The scintillation counts that were obtained for control samples of bulk milk were considerably lower than for the milks obtained from Charm Sciences, Inc. or processed using UHT pasteurization. One of 973 milk samples was suspect for tetracycline residues by means of the B. calidolactis tube test as well as by the receptor assay; 8 other samples were also considered to be positive using the receptor assay alone. The presence of tetracycline residues could not be proved for these 9 samples (residue concentration, <10 ng/ml) with HPLC. We concluded that the receptor assay was not reliable to detect tetracycline residues in raw milk at <150 ng/ml. The B. cereus test plate was determined to be an inexpensive, reliable alternative for the receptor assay for detection of tetracycline residues.
A Group-Specific Microbiological Test for the Detection of Tetracycline Residues in Raw Milk
Journal of Agricultural and Food Chemistry, 2000
The potentiality of using a luminescent Escherichia coli strain for the specific detection of tetracycline residues in raw bovine milk was investigated. The sensor cells contain a reporter plasmid carrying the bacterial luciferase operon of Photorhabdus luminescens under the control of the tetracycline responsive control region from transposon Tn10. Incubation of the cells with the sample containing tetracyclines increases the light emission of the sensor cells. The most sensitive tetracycline detection was achieved in 120 min and by using CDTA as a chelating agent in the assay. Heat-treatment of milk before the assay decreased the variations in background luminescence signals and in tetracycline-induced luminescence between different milk samples. The detection limits for tetracycline, oxytetracycline, chlortetracycline, doxycycline, methacycline, demeclocycline, and minocycline were between 2 and 35 ng/mL. Nontetracycline antibiotics did not significantly interfere with the detection of tetracyclines.
Highly Sensitive Immunochromatographic Identification of Tetracycline Antibiotics in Milk
International Journal of Analytical Chemistry, 2015
A rapid immunochromatographic assay was developed for the control of tetracycline (TC). The assay is based on the competition between immobilized TC-protein conjugate and TC in a tested sample for binding with polyclonal anti-TC antibodies conjugated to colloidal gold during the flow of the sample along a membrane strip with immobilized reactants. Conjugation of colloidal gold and the total immunoglobulin (IgG) fraction of polyclonal antibodies was used to increase the assay sensitivity to ensure low content of specific antibodies in the conjugate. This allowed effective inhibition of free TC and conjugate binding in the strip test zone. Photometric marker registration allows control of the reduction of binding, thereby enhancing detection sensitivity. The proposed assay allows TC to be detected at concentrations up to 20 ng/mL, exceeding the limit of detection of the known analogues, in a wide working range (more than two orders) of 60 pg/mL to 10 ng/mL, ensured through the use of polyclonal antibodies. The assay time is 10 min. The efficiency of the designed assay is shown to identify TC in milk; the degree of recovery of TC ranges from 90 to 112%. The precision of the concentrations measurements was no more than 10%.
Asian Journal of Dairy and Food Research, Volume 43 Issue 2: 306-312 (June), 2024
Background: This study aimed to identify tetracycline residues in milk consumed in the region of Constantine (northeast Algeria) using ELISA based tetracyclin kit. Methods: A total of 180 samples were analyzed (fresh cow milk and imported powdered milk). To compare ELISA and HPLC detection values, 22 fresh milk ELISA positive samples were confirmed by HPLC analysis. Result: 92.5% of fresh milk samples contained tetracycline residues at values between 5 and 74 µg/L and 33.3% of the samples showed concentrations between 49 and 74 µg/L, that exceed the MRLs recommended by the FDA. No significant differences (p>0.01) were found between the values obtained by the two methods.
Daily ingestion of tetracycline residue present in pasteurized milk: a public health problem
Environmental Science and Pollution Research, 2014
The objective of this study was to evaluate (qualitatively and quantitatively) the occurrence of antibiotic residue in pasteurized milk in Brazil. Pasteurized milk samples (n = 252) were collected monthly from Nov. 2010-Oct. 2011 from 21 commercial establishments (brands). A screening test (Delvotest® SP-NT) was applied to those samples. In positive (n =19) and/or suspect samples (n =24), we quantified oxytetracycline (OTC) and tetracycline (TC) by highperformance liquid chromatography with diode array detector (HPLC-DAD). OTCs were detected in all positive samples and TCs in six. In the 24suspected samples, OTCs were detected in 23 and TCs were not found in 8. Of the milk brands evaluated (n =21), the presence of antibiotic residue was not detected in 4; in the other brands, both positive and suspect samples were verified. Results indicate the presence of antibiotic residue above legal limits. According to actual milk consumption in Brazil (441 mL/kg BW/day), in only 9 of the 17 brands of milk with antibiotic residue, the estimated daily intake was at or less than the maximum recommended by the European Union. The screening test used was effective to identify the presence of antibiotic residue (OTC and TC), confirmed by HPLC-DAD. The OTC is the predominant antimicrobial used by dairy farmers. Ingestion of contaminated milk by OTC and TC can increase the resistance of microorganisms to antibiotics.
Microchemical Journal, 2008
Tetracycline (TC) antibiotics are widely used for prevention and control of disease because they inhibit the growth of bacteria. However, the presence of TC antibiotics residues in food causes harmful effects on consumer's health such as allergic reactions, liver damage and gastrointestinal disturbance, so that many countries have set MRLs (maximum residue levels). Therefore, it is necessary to detect tetracycline residues, to develop suitable analytical techniques to be used as routine screens and field detection. A new approach to the biotin-avidin mediated competitive ELISA is developed to determine tetracycline residues in milk. After optimization, the LOD and LOQ were 1.0 × 10 − 10 M (0.048 μg/L) and 1.0 × 10 − 9 M, respectively, and the working range from 3.16 × 10 − 10 M to 3.16 × 10 − 7 M toward TC in milk. No cross-reactivity was observed with the structurally similar compounds; chlortetracycline (13.7%), oxytetracycline (10%) and doxytetracycline (b 1%). Additionally percent recoveries of TC spiked in milk were quite satisfactory (∼ 90%). Comparing our results obtained in this work with others, it shows with the capability to detect TC ranging in MRLs (100 μg/L in milk) sufficiently with highly sensitivity in milk, and with simple pre-treatment. In addition, this method can apply to developing useful ELISA test kit for determination of TCs in milk.
In Vivo
Background/Aim: The presence of tetracycline (TC) and its residues in raw milk and milk dairy products poses a threat to human health due to the induction of antibiotic resistance of bacteria that can be transmitted between animals, humans, and the environment. The aim of this study was to investigate the transfer of TC from raw milk to different dairy products: pasteurized milk, boiled milk, sour milk, skimmed milk, and cottage cheese. We analyzed samples of milk from different sources: household farmers, local farms, and milk factories. Materials and Methods: The analyses of TC in milk and dairy products were performed using colloidal gold immunochromatography assay (GICA) and enzymelinked immunosorbent assay (ELISA). Results: The highest content of TC was found in the milk purchased from local household farmers; therefore, these samples were chosen for the study of TC transfer to dairy products. TC was also found in sour milk at levels comparable with those obtained in raw milk. The average TC content decreased following heat treatment of the milk, as follows: for pasteurized milk 22.07% and for boiled milk 29.35%. The highest concentrations were determined in cottage cheese in the range 200-620 μg/kg. Conclusion: TC residues are transferred from milk to dairy products in various amounts depending on the preparation conditions, and due to their chemical properties, they accumulate in concentrated derivatives, such as cheese. Therefore, TC can be identified even in cheeses prepared from milk with undetected antibiotic levels.
Journal of AOAC INTERNATIONAL, 1999
A multiresidue method for isolation and liquid chromatographic determination of oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) in milk is presented. The sensitivity of the method is adequate to meet the needs of regulatory agencies. The European Community established 100 μg/kg as the maximum residue limit (MRL) in milk for TC, CTC, and OTC. Recoveries exceeded 80% for all tetracyclines at all levels, with good precision. Correlation coefficients of standards curves for individual tetracyclines isolated from fortified samples ranged from 0.991 for CTC to 0.998 for OTC. Other antibiotics that might interfere with analysis did notinterfere with elution times of OTC, TC, andCTC. The procedure is rapid, precise, and quantitative and requires minimal preparation andminimal use of organic solvents. It can beapplied to routine surveillance programs. We canprepare 10 samples for analysis in about 1.45 h.
Chromatographic methods for tetracycline analysis in foods
Journal of Chromatography A, 1992
have served for decades as an important class of antibiotics in food animal health and production. As such, they have also been a source of concern for residue monitoring authorities around the world. In response to this concern a number of microbial inhibition, immunoassay and bacterial receptor methods have evolved for the detection of this class of compounds in various foods of animal origin. However, these methods often lack specificity and are subject to false positive and false negative results. For these reasons a number of chromatographic methods for the separation and determination of the tetracyclines isolated from foods have been developed that are capable of identifying and quantifying individual tetracycline drugs. We present here an overview of tetracycline analytical methods, including microbial inhibition, immunoassay and receptor technologies for detection, techniques for isolation from food matrices, and thin-layer chromatographic, high-performance liquid chromatographic, gas chromatographic and mass spectrometric procedures for determination of this class of compounds.
Iranian Journal of Veterinary Medicine, 2014
BACKGROUND: Tetracyclines (TCs) are broad-spectrumantibiotics that are widely used in veterinary medicine. Thepresence of TCs residues in milk is a public health concern allover the world. OBJECTIVES:This study aimed to determine TCsresiduals in pasteurized milk marketed by some dairy companiesin Tehran from April 2011 to March 2012. METHODS: 432pasteurized milk samples were purchased from supermarketssupplying the milk products of 12 major dairy companies inTehran (3 samples from each company every month), and theywere stored at -20 0C until analysis. Oxytetracycline (OTC) andTetracycline (TC) residues in each sample were extracted by aliquid - liquid phase procedure and quantitated using a highperformance liquid chromatographic (HPLC) method. Chromatographicconditions included a mobile phase as oxalic acidbuffer- acetonitril (80: 20) with a flow rate of 1mL/min and UVdetectionat 355 nm. RESULTS: TCs residuals in most milksamples were lower than 100 ppb, maximum residue level(MRL);...