关键词: G-四链体；聚合酶链式反应；滚环扩增技术；沙门氏菌；荧光信号检测

Abstract: 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 + 2.872 3 (R2 = 0.992 5) 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.

Key words: G-quadruplet; polymerase chain reaction; rolling circle amplification; Salmonella; fluorescence signal detection