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The second half of 2009 from a Swedish patient who found an almost all antibiotic-resistant \(carbapenems) antibiotic resistance. The super bacteria first discovered in India, after quickly spread to Pakistan, Bangladesh, England, Australia, Japan, has spread around the globe. Therefore, the superbugs prevention, detection, treatment is imminent. In China, fever - inflammation - to antibiotics, has formed a medical habits. According to the survey, the Chinese really need to use antibiotics in patients less than 20%, 80% belong to the overuse of antibiotics. According to statistics, China's annual 80,000 people directly and indirectly, died of overuse of antibiotics. Children under the age of 7 due to irrational use of antibiotics cause deafness number up to 30 million, accounting for 30-40% of the overall deaf children. Infections in hospital patients, the drug-resistant infection fatality rate was 11.7%, only 5.4% of the mortality of common infections. These figures make China become one of the most serious national problem of overuse of antibiotics in the world. Superbugs appear on the status quo of China's abuse of antibiotics has sounded the alarm again. To deal with infectious diseases, In addition to antibiotics, as well as traditional Chinese medicine. The mechanism of Chinese medicine treatment of infectious diseases is certainly very different from antibiotics, although different in there is not very clear. Chinese medicine may be able to become a savior. In view of this, we have to read materials in Chinese medicine as soon as possible against superbugs research directions. In this paper, we NDM-1 superbugs DNA synthesized by the method of synthesis of DNA in vitro, the NDM-1 gene Insert PUC57 plasmid, and then set up the detection of the NDM-1 gene. Then NDM-1 recombinant plasmid transformation, such as E. coli, with the containing Coptis Chinensis evaluation screening about E. coli recombinant plasmid NDM-1 super bacteria resistant in vitro model. 1 superbugs NDM-1 gene in vitro synthesized reference Genbank has announced the NDM-1 gene sequence (in Genbank. Accenssion Number: FN396876) synthetic, at both ends of the full-length 813bp NDM-1 gene plus BamHI and EcoRI enzymes cleavage sites. Design NDM-1 full-length primers, using the the OVERLAP method of formation of template DNA and then amplified by PCR method to obtain a double-stranded DNA transformation, the PCR product was then cloned into the cloning vector PUC57, transformed into DH5α E. coli and extensive amplification. The synthesis process by the Biotechnology Co., Ltd. Haibo yet complete. After PCR, enzyme digestion and sequencing confirmed that the resulting recombinant plasmid containing accurate NDM-1 gene. This plasmid course NDM-1 gene detection method can be used as a development tool. 2 NDM-1 gene quantitative PCR amplified as a standard in the previous experiment, the NDM-1 recombinant plasmid, the plasmid was extracted, its concentration detected by UV spectrophotometer, according to its molecular weight to calculate the molar concentration of known copy number of NDM-1 gene recombinant plasmid standard. TaqMan probes and primers designed according to the NDM-1 gene sequence and synthesis, the then 10-fold serial dilutions of NDM-1 gene recombinant plasmid standard, dilute the concentration of 9: 1e10,, 1e9, 1e8, 1e7, 1e6, 1e5 1e4, 1e3, 1e2copies/ml. Within-batch variance analysis for each concentration gradient of 12 wells at the same time quantitative PCR assay, the-batch difference between the analyst points 12 times, each time for each concentration gradient holes quantitative PCR; Based on the quantitative results of batch differences and intra differences. The results show that 1e3copies/ml, the lower limit of detection of this NDM-1 detection methods and good repeatability; their intra difference between 0.55% -1.43%, the difference between its batch of between 0.76% -2.41% The difference between intra batch differences are within the allowable range. Description The method of this experiment established quantitative PCR NDM-1 gene sensitivity and good reproducibility. 3 LAMP assay design LAMP primers of super bacteria NDM-1 gene NDM-1 gene sequence, and then to the front of experiments NDM-1 recombinant plasmid standard amplified amplified objects, 1e3's standard minimum concentration and deionized water as a negative control for amplification by the LAMP method. The results show the success of NDM-1 recombinant plasmid standard amplified LAMP specific bands, while the negative control, no bands, NDM-1 gene was successfully amplified LAMP method. 4 NDM-1 recombinant plasmid antibiotic resistance and the influence of the traditional Chinese medicine NDM-1 gene was cloned into the ampicillin resistance PUC57 plasmid, the recombinant plasmids were transformed into E. coli containing the NDM-1 gene, the result of the bacteria not only ampicillin resistance, and also has a kanamycin resistance, and the control without NDM-1 plasmid PUC57 plasmid only ampicillin resistance without having a kanamycin resistance described NDM- 1 gene does lead to bacterial antibiotic resistance. Next, we carried out the experimental of Coptis Chinensis suppression antibiotic resistance bacteria, Coptis decoction is applied in the antibiotic plates, then contain the NDM-1 gene recombinant plasmid engineered bacteria smear on the plate, the results show, coated with the Coptis Tablet engineered bacteria containing the NDM-1 gene recombinant plasmid, only a few colonies grow, and no plates coated Coptis large colonies grow. Huanglian containing the NDM-1 gene recombinant plasmid engineered bacteria may inhibit growth. The full text of conclusions of a successful super bacteria NDM-1 gene real-time quantitative PCR method. 2 is successfully established the LAMP detection methods of super bacteria NDM-1 gene. 3 recombinant plasmid was transformed into E. coli containing the NDM-1 gene enables E. coli antibiotic resistance, can be used as a screening model drug resistant super bacteria. 4 Chinese medicine the Coptis have significantly inhibit the role of super bacteria NDM-1 gene lead to antibiotic resistance.
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