Detection Of Food-borne Pathogens With Rolling Circle Amplification Technique

Food-borne pathogens are pathogens and bacteria can cause illness in human with food as the transmission medium. In this paper, the focus are Vibrio parahaemolyticus, Listeria monocytogenes,Shigella and Yersinia enterocolitica.The traditional method of detection of food-borne pathogens is culture method. The procedure is preliminary enrichment culture, isolation and purification with selective medium, and identification by biochemical test. The method is cumbersome,with a longer time. The accuracy and sensitivity is not very good. In order to solve this problem, Nucleotide amplification technique was used in this experiment. The specific sequence of the target bacteria was amplified, and was electrophoresed by the gel electrophoresis, to make sure that the size of amplication bands were the same as that of target bacteria sequence.At present, the mature method of nucleotide amplification is the PCR amplification technique, but the PCR amplification technique also has some drawbacks. The PCR nucleotide amplification technique requires that the target sequence must be switched between several temperature, so the PCR instrument is requisite and expensive.Rolling circle amplification technique is a new method of nucleotide amplification technique in last two years. There are some advantages of rolling circle amplification technique that is high specificity and sensitivity, isothermal amplification and high research and application value.The objective of this paper is to make full use of the characteristics of rolling circle amplification technique which applied to Vibrio parahaemolyticus, Listeria monocytogenes,Shigella and Yersinia enterocolitica detection in food. Development of a new type of rapid detection techniques of bacteria to improve and solve the problems that encountered in daily inspection.At fist, base on the principle of rolling circle amplification, specific primers and padlock probe were designed. The padlock probe is a unique technique of rolling circle amplification technique. The principle is that there is specifically binding sequence at both ends of the padlock probes, which allows the padlock probes bind with the target sequence to form a ring, then to lock both ends of the probe binding with DNA ligase, to forming a circular single-stranded DNA. Then combination a designed primer with the circular DNA, the nucleotide amplification of DNA can be carried out in just one temperature. Ultimately, we got the amplified sequence., observed the size of nucleic acid length using nucleotide electrophoresis technique to make sure the amplification is successful. If the amplified nucleotide sample containing the sequence of the target bacteria, then the detected sample is positive.The results showed that the established RCA detection system has high specificity, sensitivity and stability, which is suitable for the detection and identification of Vibrio parahaemolyticus, Listeria monocytogenes, Shigella and Yersinia enterocolitica. The detection kit was developed. Compared the kit with the traditional method, we found that the method has the following advantages:short detection time, high sensitivity, excellent accuracy, easy to carry.