利用重组酶聚合酶扩增和表面增强拉曼散射快速检测大肠杆菌耐药基因mcr-1

Rapid detection of Escherichia coli drug resistance gene mcr-1 by recombinase polymerase amplification and surface enhanced Raman scattering

中文关键词:  E. coli  耐药性  mcr-1  快速检测  重组酶聚合酶扩增  表面增强拉曼散射

英文关键词:Escherichia coli  drug resistance  mcr-1  rapid detection  recombinase polymerase amplification  surface enhanced Raman scattering

中文摘要:

      目的 利用重组酶聚合酶扩增(recombinase polymerase amplification, RPA)方法和表面增强拉曼散射(surface enhanced Raman scattering, SERS)技术建立一种大肠杆菌(Escherichia coli, E. coli)耐药基因mcr-1的快速检测方法。方法 首先, 通过RPA特异性扩增大肠杆菌耐药基因mcr-1的保守序列。然后, 以金纳米粒子(gold nanoparticles, Au NPs)作为SERS增强基底, 利用便携式拉曼光谱仪采集样品的SERS光谱。最后, 利用样品的特征SERS信号, 实现大肠杆菌耐药基因mcr-1的快速、灵敏检测。结果 样品位于505.5和840.9 cm?1拉曼位移处的SERS信号强度与其浓度的对数值之间具有良好的线性相关关系, r2分别为0.9827和0.9636。该方法的最低检测限为3.2×10?4 pg/μL, 检测时间约为30 min。结论 本研究建立的RPA结合SERS方法检测耐药基因灵敏度高、用时短, 为细菌耐药基因的快速检测提供了新思路。

英文摘要:

      Objective To establish a rapid detection method of drug resistant gene mcr-1 in Escherichia coli (E. coli) by using recombinase polymerase amplification (RPA) method and surface enhanced Raman scattering (SERS) technology. Methods Firstly, the conserved sequence of E. coli drug resistance gene mcr-1 was amplified by RPA. Then, using gold nanoparticles (Au NPs) as SERS enhanced substrate, the SERS spectra of the samples were collected by portable Raman spectrometer. Finally, using the characteristic SERS signal of the sample, the rapid and sensitive detection of the drug resistant gene mcr-1 in E. coli was realized. Results There was a good linear correlation between the intensity of SERS signal at Raman shifts of 505.5 and 840.9 cm?1 and the logarithmic value of its concentration, and R2 was 0.9827 and 0.9636, respectively. The minimum detection limit of this method was 3.2×10?4 pg/μL, and the detection time was about 30 min. Conclusion The RPA combined with SERS method established in this study has high sensitivity and short time, which provides a new idea for rapid detection of bacterial drug resistant genes.