Article pubs.acs.org/ac
hlyA Gene-Based Sensitive Detection of Listeria monocytogenes Using a Novel Cantilever Sensor Harsh Sharma and Raj Mutharasan* Department of Chemical and Biological Engineering, Drexel University, 3141 Chestnut Street, Philadelphia, Pennsylvania 19104, United States S Supporting Information *
ABSTRACT: Piezoelectric cantilever sensors are shown to exhibit sensitive and selective detection based on an identifying gene from genomic extract at ∼102−103 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 104 times excess nontarget genomic DNA extracted from E.coli JM 101 (EC), for the gene-specific detection of LM. Discernible detection limit of 7 × 102 LM cells (equivalent genomic DNA; 2.32 pg) was achieved in proteinous background. The detection limit deteriorated to 7 × 103 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. the TTR to ∼24 h while the best detection sensitivity reported to date is pre-enrichment cell population of 10−102 CFU.1a,7,11 For gene-based detection, genes such as prfA,7,12 a transcriptional activator of the virulence factor, phospholipases and internalins,13 and hlyA,1a,14 the gene that codes for the listeriolysinO toxin (LLO), have been used. The hlyA gene (Gene Accession No. X 12157) is 1717 base pairs (bp) long, and only a single copy of this gene is present in the genome of pathogenic LM. Since this gene codes for the toxin of the pathogen, it is necessary for virulence and is used for identifying LM in the presence of other Listeria strains.15 Even for the culture and plating method of detection, the last confirmation step for identifying the presence of LM involves plating LM on blood agar which shows LLO activity derived from hlyA gene. In this study, we propose the use of asymmetrically anchored piezoelectric-excited millimeter-sized cantilever (aPEMC) sensors for detection of LM via the hlyA gene sequence. We designed a detection sequence using the hlyA gene and measured hybridization on aPEMC sensors for a range of target strand concentrations. Confirmation of hybridization on the senor surface was achieved in multiple ways: (1) the use of a dye that intercalates only in double stranded DNA (ds-DNA)
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onsumption of foods contaminated with Listeria monocytogenes (LM) causes listeriosis in humans and animals. Listeria monocytogenes is found in a variety of foods such as milk, milk products, eggs, poultry, and meat.1 The FDA maintains a zero tolerance policy for LM2 since it has a low infectious dose (
