Development of rapid screening assays for the detection of foodborne pathogens in food

Fast and simple detection methods of bacteria and viruses in foodsamples have become a basic requirement in many fields, such as food production. Most rapid methods depend on immunological techniques and bio-sensing rather than the traditional culture based techniques. The purpose of this study was to develop numerous fast, simple and cheap assays to detect pathogenic bacteria and viruses in food samples and food processing plants, the methods are based on different scientific principles, such as immunological techniques with specific binding elements (antibodies and aptamers) or general capturing elements (lactoferrin), in addition to molecular methods, such as loop mediated isothermal amplification (LAMP), in the first approach, a simple and reliable colorimetric immune sensor using specific antibodies was developed and evaluated as a novel and rapid detection platform for foodborne pathogenic bacteria on surfaces of poultry processing plants, cotton swabs were activated by aldehyde groups to be used as a substrate and pre-concentration matrix for pathogens. The assay was tested on artificially contaminated surfaces with different concentrations 10[1]-10[8] CFU/ml of Salmonella typhimurium, Salmonella entritidis, Staphylococcus aureus and Campylobacter jejuni. In the second approach, the rapid detection of S. Typhimurium, S. entritidis, S. aureus,C. jejuni in addition to Norovirus was achieved using a colorimetric immune sensor, this method can be used for the on-site detection of pathogenic bacteria on the surfaces of chicken meat. In this assay, activated cotton swabs coupled with lactoferrin were used for preconcentrating pathogenic bacteria from the contaminated chicken surfaces, the color intensity of the cotton surfaces increased with the increasing concentration of the pathogenic bacteria. The detection limit was found to be as low as 10 CFU/ml for S. entritidis, 100 CFU/ml for S. entirica serovar typhimurium and C. jejuni and 1000 CFU/ml for S. aureus. This method is highly specific and was further confirmed by the LAMPmethod, the same technique was used to detect Norovirus in food samples. In the third approach, a combination of the LAMP technique and nanotechnology was used for the detection of Salmonella, E. coli. 0157H and C. jejuni in poultry processing plants. Lactoferrin was used as a cross linker between the amplified DNA sequences and with less than 50 nm containing carboxylic acid functional groups, stainless-steel surfaces were artificially contaminated with different concentrations of bacterial cultures in the range 10 to 108 CFU. Positive samples were visually detected by observing the aggregation of dyed nanospheres forming a disc near the top of solution. Conversely, negative samples were characterised by dispersed dye in the solution. This assay showed very good sensitivity, ranging between 10 CFU in both Salmonella and E.coli, and 100 CFU in the case of C. jejuni. The last approach was a fluorescence-based study for mapping the highest affinity truncated aptamers from the full-length sequence and its integration in a graphene oxide platform for the detection of S. enteritis to identify the best truncated sequence. Molecular beacons were used as well as a displacement assay design. The detection limit of the aptasensors fabricated using the truncated aptamer was lower than the full-length aptamer. Moreover, the aptasensors did not show significant cross reactivity with other related bacteria such as S. typhimurium, S. aureusand E. coli. The fluorescent/graphene oxide aptasensors also demonstrated good recovery for the detection of S. entirica serovar enteritidis from spiked milk samples.Fast and simple detection methods of bacteria and viruses in foodsamples have become a basic requirement in many fields, such as food production. Most rapid methods depend on immunological techniques and bio-sensing rather than the traditional culture based techniques. The purpose of this study was to develop numerous fast, simple and cheap assays to detect pathogenic bacteria and viruses in food samples and food processing plants, the methods are based on different scientific principles, such as immunological techniques with specific binding elements (antibodies and aptamers) or general capturing elements (lactoferrin), in addition to molecular methods, such as loop mediated isothermal amplification (LAMP), in the first approach, a simple and reliable colorimetric immune sensor using specific antibodies was developed and evaluated as a novel and rapid detection platform for foodborne pathogenic bacteria on surfaces of poultry processing plants, cotton swabs were activated by aldehyde groups to be used as a substrate and pre-concentration matrix for pathogens. The assay was tested on artificially contaminated surfaces with different concentrations 10[1]-10[8] CFU/ml of Salmonella typhimurium, Salmonella entritidis, Staphylococcus aureus and Campylobacter jejuni. In the second approach, the rapid detection of S. Typhimurium, S. entritidis, S. aureus,C. jejuni in addition to Norovirus was achieved using a colorimetric immune sensor, this method can be used for the on-site detection of pathogenic bacteria on the surfaces of chicken meat. In this assay, activated cotton swabs coupled with lactoferrin were used for preconcentrating pathogenic bacteria from the contaminated chicken surfaces, the color intensity of the cotton surfaces increased with the increasing concentration of the pathogenic bacteria. The detection limit was found to be as low as 10 CFU/ml for S. entritidis, 100 CFU/ml for S. entirica serovar typhimurium and C. jejuni and 1000 CFU/ml for S. aureus. This method is highly specific and was further confirmed by the LAMPmethod, the same technique was used to detect Norovirus in food samples. In the third approach, a combination of the LAMP technique and nanotechnology was used for the detection of Salmonella, E. coli. 0157H and C. jejuni in poultry processing plants. Lactoferrin was used as a cross linker between the amplified DNA sequences and with less than 50 nm containing carboxylic acid functional groups, stainless-steel surfaces were artificially contaminated with different concentrations of bacterial cultures in the range 10 to 108 CFU. Positive samples were visually detected by observing the aggregation of dyed nanospheres forming a disc near the top of solution. Conversely, negative samples were characterised by dispersed dye in the solution. This assay showed very good sensitivity, ranging between 10 CFU in both Salmonella and E.coli, and 100 CFU in the case of C. jejuni. The last approach was a fluorescence-based study for mapping the highest affinity truncated aptamers from the full-length sequence and its integration in a graphene oxide platform for the detection of S. enteritis to identify the best truncated sequence. Molecular beacons were used as well as a displacement assay design. The detection limit of the aptasensors fabricated using the truncated aptamer was lower than the full-length aptamer. Moreover, the aptasensors did not show significant cross reactivity with other related bacteria such as S. typhimurium, S. aureusand E. coli. The fluorescent/graphene oxide aptasensors also demonstrated good recovery for the detection of S. entirica serovar enteritidis from spiked milk samples