Establishment And Application Of New Method For Microbial Detection Based On Bio Thermodynamics

Microbal testing is not only necessary to guarantee the drug quality as well as to eliminate the clinical adverse reaction,but also it is a shortcut for seeking high-effective antibacterial drugs and solving the problem of bacterial resistence. Therefore, it has become inevitable to establish a rapid,sensitive and specific microbial detection method, in order to overcome the deficiencies of routine sterility tests such as long detecting cycle, low sensitivity and prone to misjudgments as well as the shortcomings of modern analysis technology such as expensive, complicated operation and no specificity.This research has used biothermodynamics to investigate heat metabolism along with the growth processes of organisms, using16SrDNA, MALDI-TOF MS, VITEK-2compact technology to support the identification. At the same time, the experiments of clinical verification, sterility test in injections and anti-bacteria activity evaluation have been carried out to investigate the practical value for realizing the popularization and application of the new method. The results showed that the microbial detection method based on biothermodynamics was not only rapid, sensitive and primarily traced, also couid represent the real-time, accurate and quantitative characterization of the microbial metabolism when drugs intervened.which has important application value and profound significances.The main results are as follows:1) to establish a rapid,sensitive and real-time method of microbiological testing with biological activity fingerprint.A biothermodynamics method adopted to rapidly detect the microorganism contamination of injections by monitoring the heat metabolism during the growth of microbe. The growth rate equal to or greater than zero and the heat power difference of Pi and Po with three folds higher than the noise of baseline were chosen as indexes to study the heat change rule of microbe. In this way, the effectiveness of the new method to detect strains required by conventional sterility test or in injection samples was also investigated. Results showed that the Gram-positive bacteria, Gram-negative bacteria and fungi demanded by sterility testing methodology could be detected by biothermodynamics method within10hours, with the sensitivity lower than100CFU-mL"1. Meanwhile, the same microbe in different dilution degrees had consistence in curve shape reflecting apparent finger-printing characteristics. The new method was faster and has higher sensitivity as well as potential traceability compared with the conventional one.2) The application of a noval microbial detection method based on biothermodynamics.Similarity of thermal spectrum is very high between clinical contamination samples and standard strains and fingerprints did not change with the change of dilution degrees, which had potential value for early screening of microbial contamination.In the sterility test of actual samples (Compound Yinchen injection, Shuanghuanglian powder injection and Compound Triamcinolone injection) under different sterility processing, both non-sterility samples and sub-sterility samples could be detected within lOhs. And the shorter detection time of non-sterility samples indicated its higher contamination degree. The result demonstrated that the new method has good sensitivity and feasibility towards actual injection samples.Applying microbe testing techniques of biothermodynamics, the coptisine and berberine and their combination with Imipenem and Cilastatin Sodium to Multi-drug Resistant Acinetobacter Baumarnii has been assessed respectively. Through the spectrum of biothermoactivity, the parameters has been acquired including growth rate, time of going down to posterity, peak time and max heat power, etc. And by that, the activity of anti-bacteria could be tested. The result showed that the anti-bacteria activity of coptisine is stronger than berberine and their synergistic effect with Imipenem and Cilastatin Sodium was obvious.As the results suggested above, the biothermodynamics had advantages of being fast, sensitive and cheap when applied in microbial testing method, which was promising to bring beneficial complement to other microbial testing methods. According to the study of the active components in Rhizoma Coptidis, antimicrobial activity in monomer composition and drug combination were investigated to seek a effective way,which provides references to improve the bacterial resistance and enhance the drug inhibitory ability.