hlyA gene-based sensitive detection of Listeria monocytogenes using a novel cantilever sensor.

Piezoelectric cantilever sensors are shown to exhibit sensitive and selective detection based on an identifying gene from genomic extract at ~10(2)-10(3) cells of foodborne pathogen, Listeria monocytogenes (LM). The study consists of two parts: tests with synthetic genes and experiments starting with whole LM cells. A probe designed for the virulence hemolysin gene, hlyA, was immobilized on the gold-coated sensor, and hybridization detection of a synthetic target (based on hlyA) is shown to span over 6 decades in concentration. Hybridization response was confirmed using two methods: (1) the use of a fluorescent indicator for the presence of double-stranded DNA (ds-DNA) and (2) hybridization response of a secondary single-strand DNA (ss-DNA) to the unhybridized part of the target much like in the enzyme linked immunosorbent assay (ELISA) sandwich format. Hybridization of the secondary ss-DNA tagged to gold nanoparticles amplified as well as confirmed the target hybridization to the hlyA probe on the sensor. Genomic DNA from LM was extracted, sheared, and melted and was exposed to the hlyA probe on the sensor in proteinous background with and without the presence of up to 10(4) times excess nontarget genomic DNA extracted from E.coli JM 101 (EC), for the gene-specific detection of LM. Discernible detection limit of 7 × 10(2) LM cells (equivalent genomic DNA; 2.32 pg) was achieved in proteinous background. The detection limit deteriorated to 7 × 10(3) LM (23 pg of gDNA) in the presence of genomic DNA from EC. Hybridization response times were within ~90 min, thus significantly improving over the conventional detection techniques in detection time at comparable detection limit.