Simultaneous detection of different bacteria by microchip electrophoresis combined with universal primer-duplex polymerase chain reaction

• Published in: Journal of Chromatography A 2020-03, Vol.1615, p.460734, Article 460734
• Main Authors: Luo, Feifei, Li, Zhi, Dai, Ge, Lu, Yuqi, He, Pingang, Wang, Qingjiang
• Format: Article
• Language: English
• Subjects: Duplex PCR; Microchip electrophoresis; Pseudomonas aeruginosa; Salmonella enterica serovar Typhimurium; Universal primers
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2019.460734

•MCE-based aptasensor for simultaneous detection of two kinds of pathogenic bacteria.•A novel universal primer was used in duplex PCR for nucleic acid amplification.•This method avoids the lysing of bacterial cells and simplifies duplex PCR process.•This method shows low LOD and was used in bacteria detection in defatted milk. The specific and sensitive detection of multiple pathogens is critical for the prevention and identification of health- and safety-related problems. A microchip electrophoresis/LED-induced fluorescence (MCE-LIF) method, combining an aptamer-based probe and a novel universal primer-duplex polymerase chain reaction (PCR) process (UP-DPCR), was designed to simultaneously detect two kinds of bacteria. The probe consists of a recognition unit (aptamer) for specifically capturing bacterial cells and eventually releasing complementary DNAs (C1 and C2). The two released DNA strands (C1 and C2) can be simultaneously amplified by a pair of universal primers, because of the identical sequences designed at both ends of the two DNA strands. The UP-DPCR products of C1 and C2 can be separated and detected by MCE-LIF, and the heights of the two peaks are correlated with the concentrations of the corresponding bacteria. Here, Salmonella enterica serovar Typhimurium (S. Typhimurium) and Pseudomonas aeruginosa (P. aeruginosa) were detected as a proof of concept. Under optimal conditions, the limits of detection (S/N  = 3) were 15 CFU mL−1 for S. Typhimurium and 5 CFU mL−1 for P. aeruginosa. This approach was also applied for detecting these two types of bacteria in defatted milk, indicating its potential application in the analysis of real samples. This method can not only simultaneously detect two kinds of bacteria without lysing the bacterial cells, but also simplify duplex PCR with the use of universal primers.