Sensitive and isothermal electrochemiluminescence gene-sensing of Listeria monocytogenes with hyperbranching rolling circle amplification technology

Abstract

Listeria monocytogenes (L. monocytogenes) is one of the most problematic human pathogens, as it is mainly transmitted through the food chain and cause listeriosis. Thus, specific and sensitive detection of L. monocytogenes is required to ensure food safety. In this study, we proposed a method using hyperbranching rolling circle amplification (HRCA) combined with magnetic beads based electrochemiluminescence (ECL) to offer an isothermal, highly sensitive and specific assay for the detection of L. monocytogenes. At first, a linear padlock probe was designed to target a specific sequence in the hly gene which is specific to L. monocytogenes and then ligated by Taq DNA ligase. After ligation and digestion, further amplification by HRCA with a biotiny labeled primer and a tris (bipyridine) ruthenium (TBR) labeled primer was performed. The resulting HRCA products were then captured onto streptavidin-coated paramagnetic beads and were analyzed by magnetic beads based ECL platform to confirm the presence of targets. Through this approach, as low as 10 aM synthetic hly gene targets and about 0.0002 ng/μl of genomic DNA from L. monocytogenes can be detected, the ability to detect at such ultratrace levels could be attributed to the powerful amplification of HRCA and the high sensitivity of current magnetic bead based ECL detection platform.