%0 Journal Article %T 铜绿假单胞菌环介导等温扩增技术检测方法在实验小鼠微生物控制中的应用<br>Application of Loop-Mediated Isothermal Amplification Assay of Pseudomonas aeruginosa in Microbial Control of Laboratory Mice %A 向钦 %A 王会娟 %A 刘阳 %A 伍悦 %A 曹敏 %A 赖国旗 %A 何明忠 %J 四川动物 %D 2019 %R 10.11984/j.issn.1000-7083.20180189 %X 中文摘要：建立铜绿假单胞菌 Pseudomonas aeruginosa环介导等温扩增技术（LAMP）检测方法并初步应用于实验小鼠微生物控制。根据铜绿假单胞菌 oprL基因设计LAMP特异性引物，优化反应条件，确立LAMP的检测体系；再通过对小鼠血清样本的检测，与《GB/T 14926.17-2001 实验动物 绿脓杆菌检测方法》对比，阳性结果再用PCR方法验证。新建立的LAMP方法特异性强，灵敏度比普通PCR高10 3倍；当反应温度为66 ℃，内引物和环引物的浓度分别为70 μmol·L -1和30 μmol·L -1时，LAMP反应体系最佳；利用建立的LAMP方法检测 87份小鼠血清样本，铜绿假单胞菌检出率为11.5%（10/87），比《GB/T 14926.17-2001 实验动物 绿脓杆菌检测方法》的高（0/87），阳性结果与PCR方法一致。本研究建立的LAMP方法特异性强、灵敏度高、可重复率高、稳定性好，为检测铜绿假单胞菌提供了新的研究手段。<br>英文摘要：In order to established an assay of loop-mediated isothermal amplification (LAMP) for Pseudomonas aeruginosa detection in laboratory mice. Specific primers for LAMP were designed based on P. aeruginosa oprL gene. P. aeruginosa-specific LAMP assay was established through optimizing the reaction system, and the optimal temperature of LAMP was found to be 66 ℃. The inner and loop primer concentration were 70 μmol·L -1 and 30 μmol·L -1, respectively. A total of 87 mouse serum samples were detected and compared with the standard assay of P. aeruginosa according to GB/T 14926.17-2001, and the P. aeruginosa-positive samples were further verified by PCR. Validation of the specificity and reproducibility of the LAMP assay was also performed. Finally, the limit of LAMP detection for P. aeruginosa was 10 3 times more sensitive than normal PCR assay. The detection rate of P. aeruginosa by LAMP assay in serum samples was higher than that by GB/T 14926.17-2001 assay (10/87 vs. 0/87). Therefore, we demonstrate that LAMP assay has the advantages of specificity, sensitivity, repeatability, and stability in detecting P. aeruginosa in mice. 2019,38(1): 28-36 收稿日期：2018-06-21 分类号：Q95-33 基金项目：国家自然科学基金面上项目（81570541）；重庆市科委民生项目（cstc2017shmsA130097） 作者简介：向钦(1991—),女,硕士研究生,实验动物学,E-mail:xqin0512@163.com *通信作者：何明忠,E-mail:564290461@qq.com 参考文献： 蒋观成, 浦野徹. 1991. 绿脓杆菌从小鼠肠道中的转移[J]. 中国比较医学杂志, (2): 73. 王燕, 窦恒利, 陈世敏, 等. 2010. 抗铜绿假单胞菌外膜蛋白F单克隆抗体制备及夹心ELISA检测方法的建立[J]. 细胞与分子免疫学杂志, 26(9): 880-883. 邢进, 冯育芳, 高正琴, 等. 2012. 北京地区动物实验设施动物饮水无菌检测及绿脓杆菌污染菌株鉴定[J]. 实验动物科学, 29(1): 30-33. Baker DG. 1998. Natural pathogens of laboratory mice, rats, and rabbits and their effects on research[J]. Clinical Microbiolology Reviews, 11(2): 231-266. de Vos D, Lim A Jr., Pirnay JP, et al. 1997. Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL[J]. Journal of Clinical Microbiology, 35(6): 1295-1299. Deschaght P, de Baere T, van Simaey L, et al. 2009. Comparison of the sensitivity of culture, PCR and quantitative real-time PCR %K 铜绿假单胞菌 %K 环介导等温扩增技术 %K 小鼠< %K br> %K 英文关键字： Pseudomonas aeruginosa %K loop-mediated isothermal amplification %K mice %U http://www.scdwzz.com/viewmulu.aspx?qi_id=1886&mid=58652&xuhao=4