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

目的建立铜绿假单胞菌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。方法根据铜绿假单胞菌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。结果新建立的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70μmol·L^(-1)和30μmol·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。结论本研究建立的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。

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 opr L gene.P.aeruginosaspecific 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-1and 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/T14926.17-2001,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.