PAN-FISH-based Technologies For Rapid Identification Of Foodborne Bacteria And Their Applications

Direct identification of pathogenic microorganisms from samples without cultivation is one of the ultimate goals in surveillance of foodborne pathogens and clinical microbiology.A number of techniques(such as direct staining)have been approached over the past few decades for putative identification of bacterial pathogens in suspected samples.With the development of modem molecular biology,some novel methods,such as PCR,nucleic acid probes,fluorescence in situ hybridization(FISH),have been developed as an important component of rapid diagnostic microbiology for specific detection or identification of microbial pathogens.Some of these methods have been used in clinical settings for rapid and direct identification of some clinically important pathogens.However,these methods are not suitable for rapid detection of foodbome pathogens because of complexity of the food components and low number of bacterial cells in processed foods.The food samples are often subjected to pre-enrichment to increase the bacterial population in the suspected food commodities,which would negate the efforts of rapid detection and increase the costs.Peptide nucleic acid(PNA)technology is a recent addition to diagnostic microbiology.PNA is a DNA analog composed of neutral repeating N-(2-aminoethyl)-glycine units linked by peptide bonds,which was developed in 1991 by P.E.Neilsen,a Danish scientist.It is able to bind to the complementary sequences of DNA molecules and has good chemical and biological stability.PNA has good binding affinity and rapid binding to both DNA and RNA molecules.The main objective of this study was to develop novel PNA-based methods for rapid detection of foodbome pathogenic bacteria in combination with FISH,fluorescent microscopic imaging and morphologic sizing,fluorometric scanning,and immunomagnetic adsorption.1.PNA-FISH technologies for rapid detection of major foodbome pathogenic bacteriaSolid-phase single or dual color PNA-FISH was developed for Listeria monocytogenes,Vibrio spp.,Salmonella spp.,Staphylococcus aureus,Enterobacter sakazakii and Campylobacter jejuni by using PNA probes specific to these important pathogens commonly found in foods.The probes were blasted in the PubMed database and verified in ProbeCheck.The sensitivity of the probes targeting L.monocytogenes,Vibrio spp.,and S.aureus was 100%with specificity at 99%.Optimization was conducted for different probe-bacterium combinations.The methods were specific for target bacterial species as other molecular techniques,objective by morphological features,and could be used for rapid identification on the liquid cultures or colony resuspensions.Dual-color PNA-FISH assays were further developed by optimizing combinations of the fluorescent excitation modules(I3,N21,L5 and Y3)based upon the single color format for simultaneous detection of L.monocytogenes-Vibrio,Salmonella-Enterobacter sakazakii,and Salmonella-Campylobacter jejuni.This could be conducted simply in conventional fluorescent microscope instead of using confocal microscope or flow cytometer as reported elsewhere that requires complex operational procedures and are not readily available in many common laboratories.2.PAN-FISH-based morphological identification of major foodbome bacteriaWe attempted to develop a three-layer analytical software,including the user interface layer,business logic layer and data access layer,to substitute for mere visual observation of fluorescent bacterial cells.This was achieved by constructing a database of bacterial shapes and searching-retrieving tools for morphological identification and differentiation.The system is composed of basic information of samples,shapes and sizes of target bacterial images captured,data management tools,and presentation of results as tables or figures.Differential identification is realized by comparing the fluorescent signals and bacterial images of documented species with those from the samples in terms of sizes of bacterial cells,length-width ratio,and patterns of orientation or clustering.This allows rapid,computerized and standardized identification of L.monocytogenes,Vibrio spp.,Salmonella spp.,S.aureus,E.sakazakii and C.jejuni.3.Rapid identification of Listeria monocytogenes by PNA-based molecular beaconThe solid-phase PNA-FISH requires observation on slides with fluorescent microscope,thus having limited analytical capacity.A liquid-phase PNA fluorescent scanning was developed by labeling the PNA probe with molecular beacon for rapid detection of L.monocytogenes.The 5' and 3' ends of the PNA probe were labeled with a fluorescent dye and a quencher.The labeled probe was hybridized with the fixed and permealized bacterial cells and then subjected to fluorescent scanning in a microplated-based fluorometer.The assay had high throughput for its capacity of scanning 96-wells in 10 minutes with 2-3 scanning wavelengths.It had more than 90%success rate of hybridization,a false-positive rate of 5.7-8.6%and false-negative rate of 1.4%.Therefore,the technique has advantages of time-and labor-saving over conventional fluorescent microscopic method for rapid high-throughput identification of L.monocytogenes.4.Applications of IMS-PNA-FISH in rapid detection of major foodborne bacteriaImmuno-meganetic separation(IMS)technology was combined with PNA-FISH using commercially available beads for direct identification of pathogenic bacteria in foods without pre-enrichment and primary isolation.This could avoid the bottleneck of PNA-FISH failing to detect the target bacteria directly from food samples and was used to examine L.monocytogenes and Vibrio spp.in aquatic products,Salmonella spp.and C.jejuni in meats and Salmonella spp.and E.sakazakii in milk.The technique had a lower detection limit of 100cfu/ml with significant reduction of time by two-thirds as compared with the conventional identification method.This technique had good agreements with national standardized methods for rapid identification of major foodborne bacteria except for Vibrio spp.This IMS-PNA-FISH assay could be incorporated into the computerized software system as described above for automated reporting and has the potential as alternative methods to conventional preculture/culture-purification/separation-identification procedures.In summary,the PNA-FISH technology and its liasion with morphological identification system could overcome the major drawbacks of conventional biochemical identification and several moelcular techniques based on DNA amplification.These methods,PNA-FISH and its deriving techniques,incorporate the specificity of molecular methods and visual observation of tranditional microscopic methods,the computerized and standardized image recognition system as well as the IMS technology and could serve as a novel platform for rapid and direct identification of major foodbome bacteria in foods.