Direct detection of Listeria monocytogenes DNA amplification products with quartz crystal microbalances at elevated temperatures

摘要

Current methods for identifying Listeria monocytogenes are both time and labor intensive. However, it is highly desirable to detect it rapidly and reliably to prevent and/or identify contamination of foodstuff. Herein we propose a mass-sensitive sensor operating at elevated temperatures, 50-55 °C, for that purpose. Sensitive and selective detection relies on distinguishing genes of genomic extract of L. monocytogenes. A thiol-modified ssDNA probe designed for virulence phosphatidylcholine-phospholipase C (plcB) immobilized on the Quartz Crystal Microbalance (QCM) serves as the recognition element. This hybridizes with synthetic Loop-mediated isothermal amplification (LAMP) products of target DNA on the active surface sensor. Discernible detection limits of approximately 3 × 10~(-1) to 3 × 10° CFU mL~(-1) of L. monocytogenes DMST 17303 gDNA were achieved. The QCMDNA sensor showed high sensitivity and selectivity for L. monocytogenes (100 %) with negligible interference by DNA of other foodborne pathogens, such as Salmonella Paratyphi A (24 %), Salmonella Weltevreden (24 %), Salmonella Typhi (16 %), Shigella boydii (22 %), and Shigella flexneri (13 %). The temperature covered is in the range of 50-55 °C for immobilizing DNA probe and DNA target hybridization. Hybridization response times were within 10-30 min, demonstrated by saturation of the respective sensor responses. It turned out that sensitivity of the hybridization response increases up to two times by co-immobilizing the probe and L-cysteine. The latter acts as a spacer to increase probe-probe distance. This work demonstrates the potential of the QCM sensor technique at elevated temperatures as a sensor platform for further development of sensitive, specific and rapid detection of microbial DNA.