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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
Application of Loop-Mediated Isothermal Amplification Assay of Pseudomonas aeruginosa in Microbial Control of Laboratory Mice
向钦1, 王会娟1,2, 刘阳1, 伍悦3, 曹敏1, 赖国旗1, 何明忠1*
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DOI:10.11984/j.issn.1000-7083.20180189
作者单位:1. 重庆医科大学实验动物中心, 重庆 400016;
2. 浙江大学基础医学院, 杭州 310058;
3. 重庆医科大学附属第二医院肝胆外科, 重庆 400010
中文关键字:铜绿假单胞菌;环介导等温扩增技术;小鼠
英文关键字:Pseudomonas aeruginosa; loop-mediated isothermal amplification; mice
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103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。
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 103 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
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