铜绿假单胞菌LAMP快速检测方法建立及微滴数字PCR验证

李羽翡; 祖新; 李羽翠; 张德荣

doi:10.13386/j.issn1002-0306.2020.11.041

铜绿假单胞菌LAMP快速检测方法建立及微滴数字PCR验证

Establishment of LAMP Rapid Detection Method for Pseudomonas aeruginosa and Verification of Droplet Digital PCR

摘要

摘要: 目的:采用环介导等温扩增技术建立铜绿假单胞菌快速检测方法,并用微滴式数字聚合酶链式反应(droplet digital polymerase chain reaction,ddPCR)技术进行方法验证。方法:以铜绿假单胞菌rpod基因为靶基因设计引物,建立了果汁饮品铜绿假单胞菌快速检测方法,对于方法的可靠性、特异性和灵敏度,采用ddPCR验证。结果:本实验建立的LAMP快速检测方法显色结果明显,ddPCR检测,以铜绿假单胞菌悬液浓度为横坐标,以数字PCR扩增copy数为纵坐标,生成标准曲线y=0.07497x-9.3516,线性关系良好,R²=0.9999。铜绿假单胞菌检测范围1.5×10²~1.5×10⁵ CFU/mL;方法灵敏度高、特异性强,标准菌株最低浓度为1 ng/μL;标准差分别为0.57、0.71、4.24、14.8,RSD值在0.66%~22.8%之间。检测果汁饮品128份,126份未显色,显色样品2份,以标准曲线定值,浓度分别为1.64×10⁴、2.29×10² CFU/mL。

Abstract: Objective:A rapid method for detecting Pseudomonas aeruginosa was establish by loop-mediated isothermal amplification,and verified by microdroplet digital PCR assay. Methods:Using the rpod gene of Pseudomonas aeruginosa as the target gene,a rapid detection method for Pseudomonas aeruginosa in juice drink was established by design of primers. For the reliability,specificity and sensitivity of the method was verified by the droplet digital PCR. Results:The established LAMP rapid detection method had obviously color change. In the detection of droplets digital PCR,the concentration of Pseudomonas aeruginosa suspension was used as abscissa,and the number of copy amplified by digital PCR was used as ordinate. The standard curve was y=0.07497x-9.3516.The linear relationship was good,R²=0.9999. The detection range of Pseudomonas aeruginosa was 1.5×10²~1.5×10⁵ CFU/mL The method had good specificity,high sensitivity and accuracy. The detection limit of standard Pseudomonas aeruginosa was 1 ng/μL. The standard deviation were 0.57,0.71,4.24,14.8,and the RSD value between 0.66% to 22.8%. 128 samples of juice drinks were detected,126 samples were not colored,and 2 samples were colored. The values were determined by ddPCR method,and the concentrations were 1.64×10⁴ and 2.29×10² CFU/mL,respectively.