Establishment Of PCR Assay To Detect Helicobacter Suis And Preliminary Application

According to our research group arrangements, the content of this study includes "Establishment of PCR assay to detect Helicobacter suis and preliminary application", apart from this, also includes "Cloning and purification of important proteins of Helicobacter pylori" and "Establishment of multiple real-time PCR assay to detect AIDS-associated mycoplasmas", the main contents are as follows:Part One:Establishment of PCR assay to detect Helicobacter suis and preliminary applicationTo detective and analyze the H.suis infection among420human gastric mucosa samples, which are positive in rapid urease enzyme reactions and from4hospitals locate (Chongqing Daping Hospital, The first Affiliated Hospital of Anhui Medical University, Beijing Military General Hospital and Peking University Third Hospital) in3different districts of China, polymerase chain reaction (PCR) that specializes in H.suis urease gene has been taken in this study. Design specificity primer for H.suis urease gene, amplify the corresponding target fragments, compare them with known DNA sequences and found that probability of homology is nearly100%. It demonstrates that H.suis infection among population is actually existed. The proportion of H.suis detected positive samples is6.2%(26positive of420samples). Meanwhile, infection rates might be diversity in different areas and population (0-7.7%).Beacon Designer7.0software is used for designing primer and fluorescent probe of H.suis gryB and ureB conserved regions, constructing and optimizing the real-time PCR reaction system, and evaluating its specificity and sensitivity. In this study, a rapid and accurate real-time PCR method for H.suis is established, which has higher sensitivity and less reaction time than ordinary PCR.Part Two:Cloning and purification of important proteins of Helicobacter pyloriHelicobacter pylori (H.pylori) is also a microaerophilic, gram-negative bacterium, and it can induce chronic gastritis, peptic ulcer, gastric cancer and mucosa-associated lymphoid tissue lymphoma (MALT), is listed as a Class I carcinogen. At present, the pathogenic mechanism of H. pylori infection leading to gastric cancer remains unclear.The study selects three proteins which are closely related to H. pylori infection causing gastric cancer. The porpose of the study is to clone and expression of the protein. The bioinformatics analysis incluing signal peptide and secondary structure, primer designing, polymerase chain reaction (PCR) amplification, linking to the pET28a (+) expression vector, transforming to Escherichia coli BL21(DE3), inducing expression and purifying, obtaining a large number of the three proteins, the three proteins are identified by mass-spectrum indicating that the amino acid sequences of the expressed polypeptide is100%homologous with the selected fragments of the original membrane protein. This study successfully constructs three kinds of Helicobacter pylori recombinant prokaryotic expression plasmid and the purified protein obtained in this study can provide a preliminary basis for further functional study of the protein and the pathogenic mechanism research of the H. pylori.Part Three:Establishment of multiple real-time PCR assay to detect AIDS-associated mycoplasmasMycoplasma penetrans, Mycoplasma fermentans and Mycoplasma pirum are recently discovered HIV-related mycoplasmas, and foreign studies show that these mycoplasmas are cofactors and predisposing factors of the Acquired Immune Deficiency Syndrome (AIDS). The porpose of the study is to establish a rapid, sensitive and specific multiplex real-time PCR assay for the detection of AIDS-associated Mycoplasmas. Three sets of primers and probes were designed based on the specific sequence of the ftsZ gene (M.penetrans and M.fermentans) and rpoB gene (M.pirum) and the Multiplex real-time PCR assay was established. Eight other Mycoplasmas and14pathogens were used to evaluate the specificity of the assay. The sensitivity of the assay was evaluated and compared with conventional PCR assay by using standard concentration positive plasmids. No specific amplification was presented by using22other pathogens. The sensitivity of this Multiplex real-time PCR assay was about10times higher than that of conventional PCR for M.penetrans and M.fermentans, and100times higher than M.pirum. This Multiplex real-time PCR assay was sensitive and specific, which could be used for the detection of AIDS-associated mycoplasmas and might be used in the clinical detection.