Introduction Listeria monocytogenes L monocytogenes a gram

Introduction Listeria monocytogenes (L. monocytogenes), a gram-positive bacterium and a member of the genus Listeria, is a ubiquitous and intracellular pathogen responsible for listeriosis, a fatal disease exhibiting the symptoms of abortion, neonatal death, septicemia, and meningitis. L. monocytogenes is found among pregnant women and their fetuses, immunocompromised persons, aging adults, patients with AIDS disease, cancer, organ transplant, and steroid treatment etc. with general mortality rate of 20%–30% [1–3]. Owning to its capabilities of surviving in foods under adverse conditions and of growing at low refrigeration temperatures (such as 4°C),L. monocytogenes is widely distributed in the environment and various types of foods. The consumption of L. monocytogenes-contaminated foods has been linked to major outbreaks in U.S. and Europe [4–7]. A critical step toward control and prevention of listeriosis outbreaks is to detect L. monocytogenes in environmental and food sources as rapidly as possible. A number of methodologies, including traditional culture-based methods and nucleic acid-based methods, have been developed and applied. In this review, we focused on description of the development and application of various PCR-based methodologies, including conventional, real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) and their applications in detection and characterization of L. monocytogenes and L. ivanovii in environmental and food sources. We also aim at providing a relatively complete summary of various PCR technologies combined with other technologies that can be applied, in detection, characterization and subtyping of L. monocytogenes and L. ivanovii in food and environmental sources.
PCR-based methodologies
Conventional PCR assays for detection of L. monocytogenes in environmental and food samples generally include: enrichment of L. monocytogenes present in the samples by culturing samples in a broth (e.g. Buffered Listeria Enrichment Broth (BLEB) or Brain Heart Infusion (BHI)) with selective agents and subculture to Listeria plating media; DNA extraction; species-specific detection of L. monocytogenes by target gene-based PCR; niclosamide of the PCR-amplified products on a agarose gel; and staining of the separated gel with EtBr, photograph and semi-quantitative analysis of amplified DNA bands [23].
While qPCR methodologies have been widely applied in detection of L. monocytogenes, a major problem is that PCR amplification is frequently inhibited by a variety of organic and inorganic substances present in environmental, biological, and food samples, leading to a decreased sensitivity and even totally false-negative results. The inhibitory substances may also be unintentionally introduced during transport, processing or DNA extraction steps [129]. This problem seriously limits wider applications of qPCR in detection of L. monocytogenes in environmental and food sources, calling for new methodologies. The ddPCR may provide a partial solution to this problem. The ddPCR technology is a digital PCR employing a water-oil emulsion droplet system. The “droplets” are generated in a water-oil emulsion to form the partitions capable of separating template DNA molecules. The droplets act the same role as individual test tubes/wells. ddPCR system partitions DNA samples into millions of droplets, which support PCR amplification of template molecules. After PCR, end-point PCR amplified product is detected by fluorescence intensity of probes and each droplet is analyzed or read in a flow cytometer to determine the proportions of PCR-positive droplets in original sample to determine the level of target DNA template in original sample by Poisson statistics. ddPCR workflow includes droplet generation, thermal cycling, droplet reading, and data analysis [130]. Doi et al. [131] compared the effectiveness between ddPCR and qPCR in detecting environmental DNA (eDNA) from sunfish in ponds with two different PCR reagents. They found that ddPCR had higher detection rates of eDNA in pond water than with qPCR, especially at low DNA concentrations. ddPCR displayed a detection rate higher than that of qPCR, suggesting that ddPCR is more resistant to PCR inhibitors than is qPCR. ddPCR appears to outperform qPCR for detecting eDNA (environmental DNA), especially in habitats with PCR inhibitors. Bian et al. [132] developed a novel ddPCR assay for detection of L. monocytogenes and E. coli O157. In this assay, a mineral oil-saturated polydimethylsiloxane (OSP) chip was used to overcome droplet evaporation. The droplet generation function, on-chip amplification and end-point fluorescence readout were integrated. Two different types of bacteria were simultaneously detected with differentially labeled TaqMan-MGB fluorescent probes. Compared with qPCR approach, OSP chip-based duplex ddPCR platform exhibited higher sensitivity at the level of single molecule resolution without significant cross-assay interference. Moreover, its LOD was 10 CFU/mL for both bacteria in 2h. The ddPCR was applied to quantify L. monocytogenes strains at different times during biofilm formation, and to study anti-adhesive actions of natural bioactive substances. The results showed that the approach might be applied for adhesion assays and biofilm research [133].