Study And Tentative Application Of The Detection Method For VTEC Utilizing Multiplex Fluorescent PCR

VTEC(Verocytotoxin-producing Escherichia coli), also called as SLTEC (Shiga-like toxin-producing Escherichia coli), is a group of Escherichia coli, which could produce VT1,VT2 and variation toxin that is noxious to vreo cell and Hela cell.VTEC could be transmited by food, and animal production is regarded as the main communicate source of VTEC to people, which can cause such diseases as HUS,HC,TTP to animals. VTEC has broken in most developed countries and some developing countries, and has been regarded as a menace to republic security by the world. Singapore has declared that VTEC should be examined for all animal products imported. Therefore, it is important for us to develop a rapid and exact detection method.The real-time PCR is a new detection method of nucleic acid. It is applied on bacterium, virus, and genetic disease in clinical detection.Refercing to VT1 and VT2 conservative gene, primers were designed based on the SYBR Greenâ… fluorescence PCR technology to offer reference for clinical detection and food detections by real-time PCR. Two experiments were carried out to obtain the study.Experiment 1, the construction of real-time PCR detection using SYBR Greenâ… . Based on the VT1 and VT2 conservative gene sequence, two pairs of primers were designed to develop common PCR. The electrophoresis of the PCR production indicated that only VTEC have the amplifictor of 95bp and 108bp. Through the optimized reaction system, SYBR Greenâ… fluorescence PCR detection method of VTEC has been established. The method can detect the VTEC number reaching 10cfu/mL. The test result indicated the specificity and the sensitivity of the method achieved the experimental designed request. Experiment 2, the application of the real-time PCR to detect VTEC quickly. The clinical sample was detected by the method of the experiment 1. Some samples are positive coming from the laboratory and others are unbeknown coming from collection. By this way, the applicability and sensitivity of the method to detection of VTEC sample can be tested. The test result indicated that the method can detect the VTEC number reaching to 10cfu/g after the sample culturing 6h in culture medium, and the method is fit for the meat detection and clinical detection.