| Spotted fever is a zoonotic disease caused by spotted fever group rickettsiae(SFGR)infection and transmitted by tick bites.Spotted fever is widely distributed and poses a serious threat to human health.The disease mainly causes symptoms such as fever,rash,and lymphadenopathy.Because of its clinical characteristics,it cannot be distinguished from other fever diseases.It is often aggravated by misdiagnosis and caused multiple organ damage or even death.Therefore,it is particularly important to establish specific rapid diagnostic methods for clinical laboratory testing.As a sensitive and rapid nucleic acid detection method,real-time PCR is helpful for the rapid diagnosis of Rickettsia infection in the laboratory,and it can also be used for quantitative analysis of rickettsial copy number in in vivo infection experiments.In 2013,the research team isolated a Japanese spotted fever rickettsia strain from a fever patient in the Dabie Mountains,Anhui.Based on this,further research on the pathogenicity of Rickettsia japonica is carried out,and the establishment of animal models can provide a good technical platform.This research has been carried out in two parts.The first part is to establish a real-time quantitative PCR method for the detection of Rickettsia japonica,and the second part is to construct a model of Rickettsia japonica infected BALB / c.The first part of the study was to design specific primers and probes based on the Rickettsia japonica outer membrane protein A(omp A)gene sequence,and to establish a Taq Man probe-based real-time quantitative PCR detection method for its specificity,sensitivity,and repeatability.The test was performed,and 80 blood samples collected from clinical patients were tested and compared using the established detection method and the conventional nested PCR method.The results show that the established real-time quantitative PCR method for detecting SFGR has a good linear relationship between the cycle threshold of the standard curve and the template copy number(r> 0.99),and has good sensitivity,specificity and repeatability when detecting SFGR nucleic acid samples.The second part studies the establishment of a model of rickettsial infection.First,the stored Rickettsia japonica strain was recovered,and Vero cells were infected several times to optimize the culture conditions so that they can be cloned in large numbers.The culture was collected and repeatedly purified by differential centrifugation to obtain a high-concentration rickettsial suspension,and the quantitative concentration was 3.75 × 106 copies / μl by real-time fluorescent quantitative PCR.BALB / c mice were infected by intraperitoneal injection,and the signs,weight and temperature information of the mice at 5 time points were recorded.blood and organ samples were collected at each time point which were a day 1,3,6,9,and 15.Blood cell count analysis,pathological section observation,and quantitative real-time PCR were used to detect changes in blood cells in mice after infection,pathological changes in organs observed by HE staining,bacterial copy numbers in blood organs,and tissue organ cytokine expression levels.The results showed that compared with the control group,the white blood cell count in the mouse blood cell count increased significantly on the third day,and the platelet count increased significantly on the sixth day.Weighing of organs at different phases showed that the liver and spleen weights of mice were significantly increased after infection.At the same time,combined with the HE staining results of mouse tissues,the liver showed obvious inflammatory cell infiltration on the third day of infection,and the manifold area was visible.Leukocytes aggregated,and spotted and flaky necrosis of the liver appeared on the 6th and 9th days,and hepatocyte necrosis reduced on the 15 th day;the lungs showed symptoms of a large number of inflammatory cell infiltration and alveolar wall thickening after infection.Combining with the rickettsial copy number measurement results,it is suggested that the rickettsial spreads with the blood after infection,the liver copy number reaches the peak on the 3rd day,and the copy number in the lung has always maintained a high level,corresponding to the liver at the time Both lung and lung showed obvious pathological changes.The expression levels of inflammatory cytokines IFN-γ,IL-1β,and IL-6 in organ tissues were significantly up-regulated on the third day of infection.The body’s natural immune response and continued cytokine secretion can also aggravate organ damage.The high expression of VEGF in the lungs suggests changes in vascular permeability.Combination with the increased rickettsial load,it continues to attack tissue cells.Chemokine CXCL12 activates inflammatory cells to secrete more inflammatory factors and cause abnormal immune responsesas and further aggravate tissue damage.In this study,a real-time quantitative PCR detection method based on Taq Man probe for detecting SFGR was established,which provided a fast and effective technical means for the rapid diagnosis of SFGR disease in clinical laboratories.After BALB / c mice were infected with R.japonica by intraperitoneal injection,the liver inflammatory response and interstitial pneumonia appeared similar to that of patients with Japanese spotted fever,which could be regarded as pathogenicity of Rickettsia japonica Research model. |
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