Development Of A Multiplex PCR–ELISA Detection System For Five Rickettsia Species

Objectives:Rickettsia is a kind of gram-negative prokaryotic microorganisms that parasitize exclusively in eukaryotic cells.It naturally parasites in a variety of blood-sucking arthropods and insects,such as lice,fleas,ticks,mites and so on.Rickettsioses is caused by insects infected with Rickettsia mainly through the bite or contact with its feces,which is a kind of acute febrile zoonotic disease.Due to the particularity of the transmission vector,the epidemic characteristics of the disease are regional and seasonal,but with the rapid development of tourism at home and abroad,rickettsia infection has been reported all over the world.In China,according to extensive epidemiological investigations,cases of Rickettsia infection have been found in almost 31 provinces;in addition,in recent years,new types of rickettsia have been found all over the world.The expansion of the scope of the disease and the rapid variation of pathogens indicate that Rickettsia may cause a pandemic,so Rickettsia is considered to be one of the important infectious pathogens that threaten the health of tourism.In addition,due to the influence of the war,the potential biological threat of Rickettsia still exists,so it is very important to establish accurate and effective laboratory detection and monitoring methods.In this study,a PCR-ELISA reaction model was established for multiple detection of five pathogens of Rickettsia,which provides a theoretical basis for accurate detection of Rickettsia.Methods:1.Before the experiment,a literature survey was conducted to determine the pathogens needed in this study.2.The specific and conservative gene fragments of each pathogen were selected,and the specific probes and primers were designed.In order to facilitate the experiment,all the target gene fragments of the pathogen were fused by overlap extension PCR,and the reference material of fusion gene plasmid was prepared.3.Establishment and optimization of PCR detection system of fusion gene.4.Establishment of multiple PCR-ELISA detection system and optimization of conditions.Results:1.Through the investigation of the literature,five Rickettsia were identified（Rickettsia Prowazekii、Anaplasma phagocytophilum、Rickettsia mooseri、Orientia tsutsugamushi、Coxiella burnetii）,and the target gene fragments were determined respectively,then the gene fusion was carried out and the reference material of fusion gene plasmid was constructed.In the process of fusion,the concentration of overlapping primers was optimized,and it was found that when the concentration of overlapping primers was 0.1-0.01μM,the brightness of fusion gene bands was higher and the heterozygosity was less.2.The PCR detection system of fusion gene was established,and the annealing temperature and cycle times were optimized.It was determined that the annealing temperature was 60℃and the cycle number was 32times.The specificity and repeatability of the same detection system are good.3.Establishment of multiple PCR-ELISA detection system.At the same time,the results of environmental optimization were as follows:the concentration of streptavidin was 5μg/m L,the dilution of anti-digoxin antibody labeled by peroxidase was 1:2000,the best blocking time of BSA was 2h,the hybridization temperature and time of digoxigenin labeled PCR product and biotin labeled probe were 68℃and5min,the probe concentration was 6.25μM,the binding time of hybrid product to enzyme plate was 30min,and the chromogenic time was 30min.4.The cutoff value of OD₄₅₀for multiple PCR-ELISA detection system was 0.201.In the methodological evaluation,it was found that the sensitivity of the five kinds of rickettsia was significantly higher than that of the common PCR method,and the specificity and reproducibility were good.Conclusion:Five kinds of rickettsia were detected successfully by the established multiple PCR-ELISA detection system,and the sensitivity and specificity of this method were greatly improved,so it is a multiple,timely,rapid and effective detection method.