Devenlop Of DNA Microarray For The Detection Of Seven Rickettsia

Objective:Rickettsioses are acute febrile, zoonotic diseases cased by rickettsia, which are obligate intracellular gram-negative bacteria except a very few species. At present, more than 20 pathogenic rickettsia in the word have been found. Pathogens isolated from patients and molecular diagnosis and immunology detection evidence demonstrated that there are at least 10 kinds of pathogenic rickettsiose exist in china including Murine typhus, Epidemic typhus, Q fever, Scrub typhus, spotted fever group, Cat-Scrath Disease, Human granulocytic ehrlichiosis(HGE) and Human monocytic ehrlichiosis(HME). In resent years, with a new species of spotted fever and Ehrlichia emerging, rickettsioses may sudden outbreak and spread generally. Furthermore, as potential bioterrorism pathogens, rickettsia have been attented more and more. Epidemic typhus, Q fever and Rocky Mountain spotted fever have been listed in biological warfare agent directory. Therefore, it has important practical significance to strengthen the monitoring and control of ricketesial. In this study, a chemiluminescence imaging DNA microarray method for simultaneous detection seven rickettsia was developed to provide a new high throughput detection method for clinical diagnosis and epidemiological investigation of rickettsioes.Method: First, according to the literature research results, the target pathogens and its target genes in this study are determined. Second, collect pathogens. Synthetic target genes were prepared and established by gene stitcing by overlap extension for pathogens that have no sample, and then, standard plasmid were established. Third, specific primers and probes were designed and synthesized and then screened. Fouth, the multiplex PCR reaction systems were optimized by regulating PCR system composition and the concentration ratio of primer. Fifth, positive references and negative references were prepared to evaluated the specificity of the microarray. Sensitivity references were prepared to evaluate the sensitivity of the microarray. Furthermore, we also compared the microarray with real-time PCR to evaluate the sensitivity of the microarray. Simlulated samples were prepared to evaluate the detective accuracy of blood sample.Result: First, target pathogens were determined according to literature search, which include seven rickettsia(Rickettsia prowazekii, Rickettsia mooseri, Rickettsia spotted fever, Coxiella burnetii, Orientia tsutsugamushi, Ehrlichia chaffeensis, Anaplasma phagocytophilum). Plasmid of seven rickettsia were established. Second, One universal primer and four specific primers were screened for amplification of these seven rickettsia while one universal probe and nine specific probes were screened for detection of these seven rickettsia. Third, the multiplex PCR systems were opimzed and divided into two groups. PCR system composition and the concentration ratio of primers were emphatically optimized to ensure all of the target genes were well amplified. A cost–effective chemiluminescence(CL) detection oligonucleotide microarray were for detecting Rickettsioses was established. Finally, the pivotal performance parameters of the microarray was evaluated. Specificity evaluation results indicate that the microarray can distinguish and screen these seven rickettsia. The detection sensitivity limitations of plasmid DNA and simlulated sample DNA was 1.5×102-3×103copies per reaction and 103-104copies/μl. The coincidence rate of simlulated sample detection is 100%.Conclusions : A chemiluminescence imaging DNA microarray method for simultaneous detection seven rickettsia was established successfully. This microarray provides a new high throuthput detection method for clinical diagnosis and epidemiological investigation of rickettsioes.