The Detection Of Rodent Helicobacter Infection By Multiplex Pcr And The Epidemiological Investigation

Rodent Helicobacter is a helicobacterium that is commonly found in the digestive tract of a group of rodents, mostly colonized in cecum, colon and hepatobiliary system. Until now, at least thirteen kinds of Rodent Helicobacter have been isolated from different laboratory rodents including rats, mice and gerbils. Among them, the most important ones that cause rodent helicobacter infection are Helicobacter hepaticus, Helicobacter bilis and Helicobacter rodentium.The prevalence of Helicobacter infection is common in rodents, and it exists mainly in the form of latent infection. Therefore, the studies involving rodent animal model, such as pathological study, drug screening and pharmacodynamics, will be inevitably interfered if recessive Helicobacter carriers are used. Several investigations of rodent Helicobacter infection have been reported, and the most frequently used methods to identify Helicobacter infection are isolation and culture of Helicobacter, immunological detection and PCR amplification. The purpose of this study is to establish a sensitive, reliable multiplex PCR method which can be applied to screen for Helicobacter infection among experimentally used rodents including rabbits, rats, mice, guinea pigs and golden hamster in Shanghai and the surrounding areas, which will finally contribute to the in-depth understanding of Helicobacter infection in rodents.1. Multiplex PCR assay to detect H.hepaticus, H.bilis and H.rodentium:in order to establish a sensitive, specific, rapid and efficient method to detect the most three important Helicobacter infection in experimental rodents, three pairs of specific primers were designed according to the published16SrRNA gene sequences of H.hepaticus, H.bilis and H.rodentium, and a simultaneous detection of these three Helicobacter infection was established by multiplex PCR method. The results showed that three target bands417bp,364bp and324bp can be specifically amplified by all the three pairs of primers respectively. The optimal amplification conditions are listed as the following:annealing temperature was52℃; Mg2+concentration was2.0mM; dNTP concentration was200μM; primers concentration was0.625μM.The sensitivity of multiplex PCR for H.hepaticus, H.bilis and H.rodentium was10fg. Therefore, a sensitive, specific, efficient multiplex PCR based method was established for the detection of H.hepaticus, H.bilis and H.rodentium.2. Epidemiological investigation of Helicobacter infection in experimental rodents in Shanghai and the surrounding areas:both traditional PCR and multiplex PCR test were performed to investigate the Helicobacter infection of rodents in Shanghai and the surrounding areas. The survey results showed that there are different degrees of Helicobacter infection in the rodents of Shanghai and the surrounding areas:the Helicobacter infection rates are lower in both ordinary and clean level experimental rabbit compared to other rodents; guinea pigs, rats and mice in different rearing environment have a significant difference in Helicobacter infection rate. In addition, the multiplex PCR test results showed that, in guinea pigs and rats, H.rodentium infection rate is significantly higher than the other two, and H.bilis infection is the lowest. However, in mice, there is no significant difference among the infection rates of the three Helicobacter. Finally, the large-scale investigation of different strains of rats and mice indicated that helicobacter infection rate of transgenic mice is very high, close to100%, maybe due to ineffective immunity and susceptibility to infection resulted from the normal gene sequence destroy.