THEISS PROPOSAL

A Novel Technique for the Detection of Food Borne Salmonella Strain

A Novel Technique for the Detection of Food Borne Salmonella Strain

Introduction

Salmonella is a major and important food-borne pathogen worldwide, and a leading cause of food poisoning in humans ([Gomez et al., 1997], [Tirado and Schmidt, 2001] and [Wallace et al., 2000]). Salmonella causes human gastrointestinal and other diseases by contaminated food products, especially eggs, milk, poultry, meat, water and vegetables. Increased public awareness related to health and economic impacts of food-borne contamination and illness has resulted in greater efforts to develop more sensitive, rapid and inexpensive method of pathogenic microbe detection and identification. The conventional bacteriological methods of detecting Salmonella suffer 4-6 days of non-selective pre -enrichment, selective enrichment, biochemical and serological confirmation which are too time consuming, labor intensive and costly to meet food safety control.

In recent years, Immunological methods have been successfully used to detect numbers of food-borne pathogens including Salmonella ([Aricind and Bhagwat, 2003], [Malorny et al., 2007], [Oliveira et al., 2002], [Perelle et al., 2004], [Seo et al., 2006] and [Whyte et al., 2002]). Real-time IMMUNOLOGICAL methods have shown promising results due to the rapid, sensitive and specific detection of pathogens. Unfortunately, amplification efficiencies can be different from sample to sample due to the effect caused by inhibition of amplification, human failures or preparation errors (Klerks, Zijlstra, & Van Bruggen, 2004). Although IMMUNOLOGICAL-base methods provide greater sensitivity and dramatically speed up detection, they require several operations, such as an electrophoresis step, and the risk of contamination is very real. In addition, sophisticated instruments are needed for amplification and detection, which are not always available in field labs. Therefore, a sensitive and inexpensive method needs to be developed to substitute the existing Immunologicalmethod.

A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (tyramide signal amplification in ELAST) was originally developed by Notomi et al. (2000). The principle of tyramide signal amplification in ELAST is auto-cycling strand displacement DNA synthesis in the presence of Bst DNA polymerase with high strand displacement activity under isothermal conditions between 60 °C and 65 °C within 60 min. The final amplification products are mixtures of many different sizes of stem-loop DNAs with several inverted repeats of the target sequence and cauliflower-like structures with multiple loops.

The characteristics of this method include: (I) A set of two specially designed inner primers and two outer primers recognize six distinct sequences on target DNA, so the method is expected to amplify the target sequence with high selectivity; (II) As the reaction is under isothermal condition, there is no time loss for thermal change, therefore thermal cycler is not needed. The amplification efficiency of TYRAMIDE SIGNAL AMPLIFICATION IN ELAST is extremely high; (III) Continuous amplification produces great amount of target DNA (to 109 level) as well as large amount of by products, white precipitate of magnesium pyrophosphate, within 30-60 min ([Enosawa et al., 2003], [Mori et al., 2001], [Notomi et al., 2000] and [Parida et al., 2004]), therefore, the presence of the white precipitate ...