基于G-四链体的PCR-RCA双重扩增技术检测沙门氏菌

Detection of Salmonella by G-quadruplex-Based PCR-RCA Double Amplification

将聚合酶链式反应(polymerase chain reaction,PCR)与滚环扩增技术(rolling circle amplification,RCA)联用,并把G-四链体互补序列嵌入到RCA扩增所需的哑铃环状模板上,通过PCR和RCA的双重扩增及特异性结合G-四链体的硫黄素T荧光信号增强的作用,达到基于G-四链体的PCR-RCA技术检测沙门氏菌(Salmonella)的目的。在优化的检测体系下,确定了沙门氏菌基因组DNA浓度对数与486 nm波长处的荧光信号强度具有良好的线性关系,回归方程为y=2.101x+2.8723(R^(2)=0.9925),线性范围为17 fg/μL~1.7 ng/μL。根据实验的特异性分析,表明此方法适用于沙门氏菌属的检测,对人工污染沙门氏菌牛奶样品进行检测,检出限为4.28 CFU/mL。该方法具有特异性强、灵敏度高、检出限低等优点,为实现食源性致病菌的快速检测提供新的方法。

In this study,a method to detect Salmonella was developed using polymerase chain reaction(PCR)combined with rolling circle amplification(RCA)with a dumbbell ring-shaped template into which a G-quadruplex complementary sequence was inserted.This method was based on PCR-RCA double amplification and the fact that thioflavin T(ThT)can specifically bind to the G-quadruplex,enhancing the fluorescence signal.Under the optimized detection conditions,the logarithmic concentration of Salmonella genomic DNA(x)had a good linear relationship with the fluorescence signal intensity at 486 nm(y).The regression equation was y=2.101x+2.8723(R^(2)=0.9925)in a linear range of 17 fg/μL–1.7 ng/μL.The specificity of the method was evaluated,revealing that it was suitable for the detection of Salmonella with a detection limit of 4.28 CFU/mL for artificially contaminated milk samples.This method,which has several advantages such as strong specificity,high sensitivity and low detection limit,provides a new method for the rapid detection of foodborne pathogens.