| Today, science and technology changes with each passing day, molecular biology and microelectronics technology has rapidly developed. New technology and new methods are rapid, accurate, specific test microbes are constantly emerging. Microbiological technology which has been from culture level to molecular level in the development direction of instrumentation, automation, standardization, has improved the efficiency, accuracy and reliability in terms of microorganism inspection.But in the field of Chinese drug inspection, microbial identification technology is very few. As the changes of the environment, strains constantly are going on genetic variation.When control strains had been inspected in accordance with the standard on, cycle was long, sometimes false negatives appeared. Gradually at present, The People’s Republic of China pharmacopoeia standards with international economy gradually will be achieved coordinated with the European pharmacopoeia, the United States pharmacopoeia and the Japanese Pharmacopoeia that appropriate appraisal method is confirmed in the aspect of control bacteria identification.Pseudomonas aeruginosa is not only one of the important pathogens of clinical common but also an important control bacteria in pharmacopoeia records of local drug, which has a high mortality. It is easy to colonize, variation and multiple drug resistance easily. They widely exists in the soil, water, air and human skin, respiratory tract and intestinal tract. So far, the bacterium is drugs, cosmetics, as well as the experimental animal inspection an important indicator in microbial control.Normal samples generally need be treated with sterilization, residual strains are more or less with varying degrees of damage to the degree which can be detected to the limination of recovery to be checked out in some medium, which certainly will need to conform to the requirement of the high sensitivity, short initial time, high concentrations of bacteria.Due to the limitation of testing instruments, if more experimental data will be get, inspection should be extremely cumbersome, which is a bottleneck problem in the class of microbiology experiment. Traditional identification method is increasingly exposed the shortcomings, which is not adapted more and more to fast-food consumption concept from the consumers. The pursuit of rapid and simple detection at the same time, the accuracy of the results will be more needed.This article to pseudomonas aeruginosa typical representative for the control of bacteria, has been a focus on exploring the most suitable liquid enrichment culture medium and culture conditions. Nine positive strains from different sources and 16 kinds of culture media at home and abroad has been choosed, OD value which was drawn the growth curve has been measured by ELIASA,which has greatly reduced the workload and become at the forefront of the enrichment medium filtering technology. At the same time, the above 9 strains would been further explored by biochemical identification and classification methods combined with the biochemical identification technology and molecular classification technology widely used.Purpose: With the method of merit, liquid enrichment culture medium of high quality, high sensitivity has been selected in some culture conditions. By traditional identification method, the international gold standard of technology, the fluorescence detection and genotyping, implicit residual factors have been found in the inspection process by the most direct, the most economic appraisal and genotyping method,which will provide a reference for pharmaceuticals, cosmetics and experiment animal testing.Methods: Liquid enrichment culture medium and culture conditions has been screened by the parameters from the growth curves, the concentration of pyocyanin and biofilm growth situation. Fresh strains have been identified though the traditional method, the API reagent,VITEK analysis system identification, serotyping and fluorescence serotype quantitative PCR identification and RAPD genetic classification. This paper have analyzed the reasons of residual.Result: TSB culture medium is the most conform to the requirements of the quality of a liquid medium, and small differences in each production unit at home and abroad provided quality products. Under the condition of 35 ℃the time to develop the most appropriate is 48 hours. The longer the appropriate extension enrichment time the higher positive detection rate. It can be extended to 72 hours if it is necessary.Traditional identification method, the article API reagent, VITEK 2 compact type automatic analysis system identification, serotyping and fluorescence quantitative PCR identification, positive detection rate was 100%,RAPD genotyping, positive detection rate was 0%.Conclusion: TSB culture medium screened, in a certain culture conditions, sensitivity is higher, the initial enrichment time is shorter, concentrations of bacteria, is higher, which is the most suitable for enrichment of pseudomonas aeruginosa.Using traditional identification method, sometimes phenotypic variation strains to varying degrees encountered, which make pyocyanin or velum produced come and go, would cause biochemical changes in the test to identify the project. Thus rely on pyocyanin or biofilm alone has been decide whether further trials of biofilm would be go on may be somewhat one-sided. Article API reagent and VITEK 2 compact type full-automatic microbial analysis system have identified all positive strains. But article API reagent readings influenced by artificial factors. While the latter has avoid the personal error, appraisal result is more reliable.Using immune serum, many factors affect the reliability of the results, in addition to sensitivity, fungus age, strain pure degree, the tool material and clean degree and so on. Results are poor repeatability and complicated operation.The reliability of the immune serum to identify is more influence factors, poor repeatability, complex operation. RAPD genotyping technology must provide a higher purity DNA sample, the fluorescent quantitative PCR identification instrument is higher sensitivity in contrast. Fluorescence quantitative PCR technology also need extracted DNA samples in this experiment. Under the circumstances directly through the pyrolysis product and in the presence of other similar strains, specificity is needed further improvement.Even under the same sample, the test result of the manufacturer to provide and I make some discrepancy, in addition to operating error, and reagent adding proportion, reagent itself also has a lot to do with the physical and chemical properties and the sensitivity of PCR instrument. |
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