The Studies On Rapid Test Kit For β-lactamase

β-lactamase was produced and secreted by gram positive bacterium, which wasusually called “antibiotic destroyer”in raw milk. It can destroy the β-lactamantibiotics in raw milk optionally. Penicilloic acid（the decomposer of penicillin Gpotassium by β-lactamase） has the acidity and reducibility so that it was detected byiodometry method.β-lactamase was produced and secreted by gram positivebacterium, which was usually called “antibiotic destroyer”in raw milk.It can destroythe β-lactamse antibioti in raw milk optionally. Penicilloic acid（the decomposer ofpenicillin G potassium by β-lactamase） has the reducibility and acidity so that it wasdetected by iodometry method,acidometry method, HPLC and spectrophoto-metrymethod. The minimum test limitation1. The standarded β-lactamase and the decomposer of Antibiotics in raw milkwas studied contrastively by SDS-PAGE electrophoresis method. Throughiodometry method, the activity of standarded β-lactamase was determined24.552×10⁴U/mL, while the decomposer of Antibiotics in raw milk23.808×10⁴U/mL. The characteristic peak m/z373was not obvious and the peakm/z333and m/z289were not observed, while penicilloic acid characteristic peakm/z307.0was obvious from mass spectrum diagram of penicillin potassium.According to mass spectrum diagram analysis, the mass in products of penicillin inβ-lactamase conditions was penicilloic acid. The penicilloic acid raised obviousiywith the increasing of the β-lactamase through HPLC method and R²0.9331, so thepositive correlation between β-lactamase and penicilloicacid was definitely.2.The penicilloic acid could reacted with iodine while the penicillin Gcouldn’t. The qualitative analysis of β-lactamase could be made by the color fadingof the iodine solution in the process of the direct iodometric method. Another side,quantitative analysis could be made by detecting the β-lactamase in milk throughindirect iodometric method and R²was0.9895. The actual minimum test limit was15U/mL. Moreover,the penicilloic acid possess acidity,the correlation curve betweenthe pH value and the β-lactamase was made through acidometry method, as R²was0.9917. The actual minimum test limit was12.3U/mL. 3.The penicilloic acid has reducibility and it can reduce copper（Ⅱ） intocopper（Ⅰ）.The2,2’-biquinolyl can coupling with copper（Ⅰ）.The amaranthcomplexes was scanned from wavelength400nm to900nm by spectrophotometer.The complexes had a maximum absorption peak in547nm, while its color depth andbeta-lactamase concentration had positive correlation. According to absorption peaksat547nm and the optimization experiment, it was determined in the optimalconditions. The optimal reaction conditions were the color developing time15min,concentration of substrate50U/mL,0.05%2,2’-Biquinoline2.4mL,20mmol/mLCuSO40.2mL and0.1mL of sample. The linear relationship was established, whichR²was0.9936and the relevance was very good. Its theory minimum test limit was3.44U/mL while the actual minimum test limit was4U/mL. The reducing reagent ofascorbic acid does not affect the testing result in the anti-interference test.4. The rapid test kit of β-lactamase was designed in the basis ofspectrophotometry test. Kit consised of reagents, comparison tubes and standardcolourimetric card.Choosing eight sample in the rang of0to200U/mL, the standardcolourimetric card was made in the optimal reaction conditions. Reagent A is TCA of13%. Reagent B is2,2’-biquinolyl of0.05%. Reagent C is CuSO₄of20mmol/mL.Choose the optimum conditions when tested sample.The optimal reaction conditionswere the developing time15min,concentration of substrate50U/mL,20mmol/mLCuSO₄0.2mL and0.1mL of sample. After15min color developing time,contrast thecolor of sample with the standard colourimetric card and detecte the consistence ofreagents. Comparing the rapid test kit, iodometry method, acidometry method,HPLC and spectrophotometric method, it can be found that the rapid test kit has highsensitivity, operation simple, economic and practical advantages.