547 Fluorescence Biosensor for a Simple and Rapid Detection of Escherichia coli in Fresh-Cut Fruits P. Chaumpluk 1, a and P. Chaiprasart 2 1 Laboratory of Plant Transgenic Technology and Biosensor, Department of Botany, Faculty of Science, Chulalongkorn University, Phyathai, Bangkok 1033, Thailand 2 Postharvest Innovation Center, Faculty of Agriculture, Natural Resource and Environment, Naresuan University, Phitsanulok, 65000, Thailand Keywords: Escherichia coli detection, biosensor, DNA sensor, fluorescence Abstract Pathogenic microorganisms associated with fresh-cut produce can cause disease outbreaks, thereby demonstrating the need for quality and safety monitoring efforts to control risks associated with these products. We have developed a rapid Escherichia coli detection method for fresh cut fruit based on loop-mediated isothermal DNA amplification with fluorescence signal detection upon binding of the target DNA products with minor groove binder. Detection processes were divided into two steps. The first was an enrichment procedure that used fresh cut mango and papaya to enable DNA amplification without any sample pretreatment such as full step DNA extraction. The second was a specific DNA amplification of the malB gene at 65°C isothermal temperature and DNA signal detection. DNA signals were measured using fluorescence visualization. The limit of detection for the method was 5 copies of E. coli DNA per 50 g of sample. No cross-reactivity was observed from samples contaminated with other bacteria. Detection could be completed within 4 hours of initial sampling, including the enrichment process, without the need of a thermo cycler. This method constitutes a basis for a rapid yet simple detection of pathogenic bacteria suitable for field application. INTRODUCTION Products of fresh-cut fruit have become a high-demand item in the retail and food service industries as a result of changes in consumer’s life style and dietary habits, demands for healthier diet, convenience and quality serving. That increasing demand has accelerated changes in production and processing methods, in sourcing of produce, and the emergence of pathogens in raw agricultural commodities that are grown outdoors. All these changes are linked to a potential increased exposure of the general public to fruits and vegetables that could be contaminated with human pathogens. Escherichia coli is one of the most dangerous food-borne pathogens and ingesting contaminated food can be deadly. It is therefore critically important to accurately and rapidly detect these potential infections. The standard process for E. coli detection in food involved either isolation of the pathogen followed by culture on the selective media or serological testing. These take more than 24 h and rely heavily on laboratory facilities (Rompré et al., 2002). Although these techniques are practical and reliable, they are time consuming and laborious (Moon et al., 2004). The recently introduced polymerase chain reaction (PCR) has been demonstrated to reduce testing time to several hours. Several PCR based detections had been demonstrated (Yuan et al., 2009; Takahashi et al., 2009). None has proven to be applicable at field sites because of their technical limitations in amplification of nucleic acid involving with a thermal cycler and hands on technical experiences. Moreover, detection generally needs an electrophoresis step to detect target DNA amplicons, which can take several hours. To reduce those limitations, a combined technique of a novel nucleic acid amplification method called Loop-mediated isothermal amplification (LAMP) and DNA detection via a fluorescence based DNA binder was used. LAMP relies on a self cycling a piyasakcha@gmail.com Proc. IS on Postharvest Pacifica 2009 Eds.: E.W. Hewett et al. Acta Hort. 880, ISHS 2010