基于环介导等温扩增方法高效检测模拟眼内液中铜绿假单胞菌的研究

Efficient detection of Pseudomonas aeruginosa in simulated intraocular fluids based on loop mediated isothermal amplification

目的建立一个稳定、高效检测眼内液中铜绿假单胞菌的体系。方法采用环介导等温扩增(LAMP)技术,由0.1~0.2 mL眼内平衡盐溶液(BSS)模拟眼内房水或玻璃体,利用铜绿假单胞菌gbca基因,特异性设计5组LAMP引物,并对扩增方法进行关键试剂的优化,检测眼内液中铜绿假单胞菌。结果该扩增方法经过优化后,最低可以检出100 fg/μL纯基因组DNA,接近荧光定量灵敏度,具有很好的重现性。此外该方法在检测眼内液中混合的菌落时呈现出良好的检测效果,可以检测出2.1×10^2 CFU/mL,极具应用价值。结论建立了一个稳定、高效、灵敏的恒温扩增体系,可以应用于眼内液中铜绿假单胞菌的检测。

Objective To establish a stable and efficient system for the detection of Pseudomonas aeruginosa in intraocular fluids.Methods Loop mediated isothermal amplification(LAMP)was applied.Balance salt solution(BSS)0.1~0.2 mL was used to simulate intraocular aqueous humor or vitreous.Five groups of LAMP primers were specifically designed by using the gbca gene of Pseudomonas aeruginosa,and the key reagent was optimized to detect Pseudomonas aeruginosa in intraocular fluids.Results After optimization,the amplification method could detect 100 fg/μL pure genomic DNA at the minimum,which was close to the fluorescence quantitative sensitivity and had good reproducibility.In addition,this method presents a good effect in the detection of bacterial colonies mixed in intraocular fluid,and can detect 2.1×102 CFU/mL,which is of great application value.Conclusions It establishes an efficient,stable and sensitive isothermal amplification system that can be applied to the detection of Pseudomonas aeruginosa in intraocular fluids.