L. monocytogenes and Campylobacter spp. are two important foodborne pathogens. They can be acquired by ingestion of contaminated food mainly ready to eat (RTE), undercooked chicken, and milk and dairy products respectively. The symptoms are gastrointestinal disorders that can switch in a serious disease like listeriosis and campylobacteriosis in weak individuals. The currently recommended ISO standard methods are sensitive and ensure compliance with microbiological criteria, but require long times and a lot of work. In order to avoid recalls or economic losses, food industries need rapid protocols that can provide results in short times. In this thesis, with the perspective to develop rapid and efficient molecular detection methods, species-specific primers and probes were designed for L. monocytogenes and C. jejuni, C. coli, C. lari, and C. upsaliensis. For the detection of L. monocytogenes in cold-smoked salmon samples and ham factory samples designed MAR1- MARB primers were applied in PCR and qPCR protocols. Two probes, ListCapt and ListE, were tested for the detection of Listeria monocytogenes. The ListCapt probe was applied on a DNA-biosensor based on the organic electrochemical transistor (OECT), after preliminary optimization tests with dot blot assay. Instead, the ListE probe was applied on a DNA- electrochemical biosensor based on voltammetry. Samples of cold-smoked salmon (CSS) and Ham factories samples (from food and environment) were analyzed by electrochemical biosensors. In parallel to molecular methods, AFNOR validated Listeria Precis™ method (ISO standard equivalent method) was applied to the food samples, to compare traditional plate count methods to molecular techniques, in both traditional and molecular approaches. L. monocytogenes was detected in only one of the tested CSS samples. Instead, six samples from ham factories were positive to the presence of L. monocytogenes. Both PCR and qPCR protocols allowed the detection of L. monocytogenes, confirming the capability of primers to detect the pathogen from a complex matrix. However, an enrichment step of 24 h was necessary. After dot blot protocol optimization to assess specificity and sensitivity of oligonucleotide probes were employed to evaluate the development of a DNA-electrochemical biosensor based on OECT and voltammetry. The studies have been made with promising results, anticipating the prospective potential of the system for label-free DNA sensing. For the detection of Campylobacter spp. designed primers CampyP were applied in PCR and qPCR protocols in 20 chicken meat samples. The probe CampyP3 was tested for the detection of Campylobacter spp. and was applied to a DNA-electrochemical biosensor based on voltammetry. Chicken meat samples were analyzed by the electrochemical biosensor. In parallel to molecular method, ISO 10272-1:2006 was applied to food samples, to compare traditional plate count methods to molecular techniques. With both traditional and molecular approaches, Campylobacter was detected in five tested samples. Both PCR and qPCR protocols allowed the detection of Campylobacter spp., confirming the capability of primers to detect the pathogen matrix without enrichment for the application of qPCR technique. After dot blot protocol optimization, to assess specificity and sensitivity CampyP3 was employed to evaluate the development of a DNA-electrochemical biosensor based on OECT and voltammetry. After the optimization, some food samples were analyzed and confirmed the data obtained by ISO and qPCR.

DEVELOPMENT OF AN ELECTROCHEMICAL BIOSENSOR FOR FOOD SAFETY: DETECTION OF FOOD PATHOGENS / Priya Vizzini , 2020 Apr 21. 32. ciclo, Anno Accademico 2018/2019.

DEVELOPMENT OF AN ELECTROCHEMICAL BIOSENSOR FOR FOOD SAFETY: DETECTION OF FOOD PATHOGENS

VIZZINI, PRIYA
2020-04-21

Abstract

L. monocytogenes and Campylobacter spp. are two important foodborne pathogens. They can be acquired by ingestion of contaminated food mainly ready to eat (RTE), undercooked chicken, and milk and dairy products respectively. The symptoms are gastrointestinal disorders that can switch in a serious disease like listeriosis and campylobacteriosis in weak individuals. The currently recommended ISO standard methods are sensitive and ensure compliance with microbiological criteria, but require long times and a lot of work. In order to avoid recalls or economic losses, food industries need rapid protocols that can provide results in short times. In this thesis, with the perspective to develop rapid and efficient molecular detection methods, species-specific primers and probes were designed for L. monocytogenes and C. jejuni, C. coli, C. lari, and C. upsaliensis. For the detection of L. monocytogenes in cold-smoked salmon samples and ham factory samples designed MAR1- MARB primers were applied in PCR and qPCR protocols. Two probes, ListCapt and ListE, were tested for the detection of Listeria monocytogenes. The ListCapt probe was applied on a DNA-biosensor based on the organic electrochemical transistor (OECT), after preliminary optimization tests with dot blot assay. Instead, the ListE probe was applied on a DNA- electrochemical biosensor based on voltammetry. Samples of cold-smoked salmon (CSS) and Ham factories samples (from food and environment) were analyzed by electrochemical biosensors. In parallel to molecular methods, AFNOR validated Listeria Precis™ method (ISO standard equivalent method) was applied to the food samples, to compare traditional plate count methods to molecular techniques, in both traditional and molecular approaches. L. monocytogenes was detected in only one of the tested CSS samples. Instead, six samples from ham factories were positive to the presence of L. monocytogenes. Both PCR and qPCR protocols allowed the detection of L. monocytogenes, confirming the capability of primers to detect the pathogen from a complex matrix. However, an enrichment step of 24 h was necessary. After dot blot protocol optimization to assess specificity and sensitivity of oligonucleotide probes were employed to evaluate the development of a DNA-electrochemical biosensor based on OECT and voltammetry. The studies have been made with promising results, anticipating the prospective potential of the system for label-free DNA sensing. For the detection of Campylobacter spp. designed primers CampyP were applied in PCR and qPCR protocols in 20 chicken meat samples. The probe CampyP3 was tested for the detection of Campylobacter spp. and was applied to a DNA-electrochemical biosensor based on voltammetry. Chicken meat samples were analyzed by the electrochemical biosensor. In parallel to molecular method, ISO 10272-1:2006 was applied to food samples, to compare traditional plate count methods to molecular techniques. With both traditional and molecular approaches, Campylobacter was detected in five tested samples. Both PCR and qPCR protocols allowed the detection of Campylobacter spp., confirming the capability of primers to detect the pathogen matrix without enrichment for the application of qPCR technique. After dot blot protocol optimization, to assess specificity and sensitivity CampyP3 was employed to evaluate the development of a DNA-electrochemical biosensor based on OECT and voltammetry. After the optimization, some food samples were analyzed and confirmed the data obtained by ISO and qPCR.
21-apr-2020
BIOSENSOR; NPS; PATHOGENS; FOOD
DEVELOPMENT OF AN ELECTROCHEMICAL BIOSENSOR FOR FOOD SAFETY: DETECTION OF FOOD PATHOGENS / Priya Vizzini , 2020 Apr 21. 32. ciclo, Anno Accademico 2018/2019.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11390/1185476
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