Generation of aptamer against Salmonella serovar enteritidis and development of aptamer-based capillary biosensor

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Tezin Türü: Doktora

Tezin Yürütüldüğü Kurum: Orta Doğu Teknik Üniversitesi, Fen Edebiyat Fakültesi, Biyolojik Bilimler Bölümü, Türkiye

Tezin Onay Tarihi: 2014

Öğrenci: CEREN BAYRAÇ

Danışman: HÜSEYİN AVNİ ÖKTEM

Özet:

Food poisoning became one of the most important diseases that threaten to human health. Among all the food-borne pathogens, Salmonella is the most common cause of food-borne infectious disease in the world. Among over 2500 serovars, Salmonella enterica serovar Enteritidis is a common foodborne pathogen associated with human diseases. Limited and time consuming diagnostic techniques such as culturing method or polymerase chain reaction lead to increase in the demands of new detection methods. Therefore, in this study we used Cell-SELEX to find DNA aptamers that bind and recognize S. Enteritidis as a sensing element and applied them to construct aptamer based sandwich type detection platform. Selection was finished at round 12 of SELEX and three sequences were determined as candidate aptamers for further studies. Among them, crn-1 and crn-2 showed high specificity and affinity against S. Enteritidis with Kd of 0.971 x 10-6 M and 0.309 x 10-6 M, respectively. The construction of aptamer based sandwich type platform was optimized with the use of S. aureus aptamer (SA-31). The optimum parameters were found as 10 µM capturing aptamer, 10 µM signaling aptamer, washing at 10 µL/sec injection speed and 5% of BSA as surface blocker. Aptamers for S. Enteritidis were then applied to platform with these optimum parameters. The detection limits of crn-1 and crn-2 based platforms were 103 CFU/mL. The response of detection platforms were evaluated for samples at 4ºC and 37ºC. It was observed that crn-1 based platform gave responses only at 4ºC, while crn-2 based platform responded at 4ºC and 37ºC.