Ultrasensitive detection of pathogenic bacteria by primer exchange reaction coupled with PGM

Abstract

This study proposes an innovative approach for highly sensitive and selective detection of pathogenic bacteria based on primer exchange reaction (PER) coupled with a personal glucose meter (PGM). The modified nucleic acid capture probe (CP, 3ʹ-end modified biotin) was immobilized onto the surface of magnetic beads (MBs) and hybridized with the specific aptamer of Escherichia coli O157:H7 (AP) to form a double-stranded structure on the surface of MBs. The AP preferentially bound to E. coli O157:H7, thereby exposing some CP. Further, CP hybridized with a 5ʹ-end sequence of long single-stranded DNA, which was autonomously synthesized by PER, and captured a nucleic acid reporter probe (5ʹ-end modified biotin) via nucleic acid hybridization. Subsequently, numerous SA-labeled invertase conjugates were added. Next, glucose was produced on adding the substrate (sucrose) of the invertase, which was detected using PGM. Under optimal conditions, the experimental results showed the corresponding detection of E. coli O157:H7 with limits as low as 10 CFU/mL and linear ranges 10–107 CFU/mL. The proposed biosensor was used to detect pathogenic bacteria in milk samples successfully.