| Budget Amount *help |
¥6,300,000 (Direct Cost: ¥6,300,000)
Fiscal Year 1998: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1997: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1996: ¥3,300,000 (Direct Cost: ¥3,300,000)
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| Research Abstract |
Many antimicrobial agents are used worldwide for the prevention and treatment of infectious diseases in animals. With regard to the detection of residual antimicrobial agents from foodstuffs, there have been many reports of methods such as thin-layer chromatography and high-performance liquid chromatography However, these methods require a long time for judgment of the residue. Therefore, the development of a simple and rapid laboratory detection method is desired at inspection sites. Antimicrobial agents can be grouped according to their chemical structure, such as amino groups (-NH2) or carboxyl groups (-COOH). We tried to develope a simple direct screening using a two-beam fluorescence detector with a fiber unit system, for some antimicrobial agents possessing animo groups.
Aampicillin (ABPC), amoxicillin (AMLPC), streptomycin (SM), dihydrostreptomycin (DSM), kanamycin (KM) and sulfamethazin (SMT) were selected Fluorescamine (Sigma) was used as a fluorescent labeling reagent for amin
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o groups. The double-beam fluorescence detector and fiber unit system emplayed was a CAM-230L (JASCO Co., Tokyo). This fiber unit has a small vinyl tube to fiber lens for sample injection, and the fiber is cylindrical, measuring 5 mm phi x 30 mm long. Pijucipally, after mixing each drug with fluorescaniine (100 mug/ml) of 10 mug/ml concentration, the excitation and emission wavelengths (nm), and minimal detectable concentration (mug/ml) were examined in a daik box (20 x 30 x 15 cm). Next, using minimal medium (MM) with Bacillus subtilis ATCC-6633 as a test organism. The inhibition zone was produced on the MM by the paper disc (10 mm in diameter) method using 2-fold dilutions (100-0.01 mug/mI) of antimicrobial agents. Then, the fluorescamine solution was spotted on the clear inhibition zone through a vinyl tube, and the fluorescence intensity was measured by the two-beam fluorescence detector and fiber unit system.
The most suirable excitation and emission wavelength for all drugs were 394 nm and 480 nm, respectively The most suitable reaction time after mixing the drug and fluorescamine solution was about 5 mm. The fluorescence intensity for each dilution of the standard drug increased gradually with the lower concentration. However, it was difficult to identify each drug by its fluorescence intensity The detection limit of each drug using the fluorescence detector was almost the same as that of the detectable inhibition size on MM medium. The limits of ABPC, AMPC, SM.DSM, KM and SMT were 0.1, 0.1, 3.13, 6.25, 0.78, 3.13 mug/ml, respectively, and the fluorescence intensity (more than 57) was strong compared with blank MM medium. In this experiment, only standard solutions of drugs possessing amino groups and an inhibition zone on MM medium were examined. It was clarified that it was possible to directly detect antimicrobial agents with amino groups using this fluorescence detector. hi the future, using meat, egg and milk samples fortified with drugs, further studies aimed at developing a new rapid screening method for residual antimicrobial agents by double-beam fluorescence detection with a fiber unit system should be done. However, it is difficult to measure the clear fluorescence intensity according to the smaller size of the inhibition zone. Less
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