| Ticks were important medical arthropods,which can absorb the host blood to transmit pathogens.They were the second largest vector of infectious diseases after mosquitoes.Ticks are widely distributed in China and can spread a variety of pathogens,mainly including spotted fever group rickettsia,Anaplasma,Borrelia burgdorferi,Babesia and so on.In recent years,with the in-depth study of ticks and tick-borne pathogens,a variety of emerging tick-borne pathogens have been found in China,which were pathogenic to humans.The public health problems caused by them can not be ignored.Therefore,it was of great significance for the prevention and control of tick-borne pathogens to master the prevalence of emerging tick-borne pathogens and to detecte emerging tick-borne pathogens quickly,with high sensitivity and high specificity.Objective1.To establish rapid,highly sensitive and highly specific fluorescence quantitative PCR methods for the detection of tick-borne pathogens.2.The epidemiological investigation of tick-borne pathogens and the study of co-infection of pathogens in the sampling area was carried out to find emerging tick-borne pathogens.The evolutionary status of tick-borne pathogens was determined by sequence alignment and phylogenetic analysis,to master the prevalence of emerging tick-borne pathogens in the local area.Methods1.MEGA7.0 software was used to compare multiple gene sequences of Spotted Fever group rickettsiae,Anaplasma,Borrelia burgdorferi and Babesia,respectively,to screen the conserved regions.The omp A gene of Spotted Fever group rickettsiae,16Sr RNA gene of Anaplasma,fl A gene of Borrelia burgdorferi and 18Sr RNA of Babesia gene were selected as the target genes for the design of primers and probes.Oligo7.0software was used to design primers and probes for the conserved regions of the pathogens.Four single fluorescence quantitative PCR methods for the detection of tick-borne pathogens were constructed,and the concentration of primers and probes was optimized to determine the optimal working concentration of primers and probes.SAS9.0 software was used to compare the amplification efficiency of primers and probes with different concentrations,and then the limitation of detection and cross-reactivity of the detection methods were determined.Based on the constructed four single tick-borne pathogens fluorescence quantitative PCR detection methods,two double tick-borne pathogens fluorescence quantitative PCR detection methods were constructed.The limitation of detection of the methods was evaluated.2.Ticks and host animals were collected from Jingxi City,Guangxi Province,Zhoushan City,Zhejiang Province,Xilingol League,Inner Mongolia Autonomous Region and Mudanjiang City,Heilongjiang Province in China from March to May 2021,and the collected samples were detected by the successfully constructed double quantitative fluorescence PCR systems for tick-borne pathogens.3.The specific target genes of the positive samples were amplified by PCR.After sequencing of the amplified products,double-terminal splicing was performed using CLC Genomics Workbench software.The genetic similarity with known pathogens was confirmed through BLAST tool.MEGA7.0 software was used to construct phylogenetic tree and determine the evolutionary status of the pathogens.Results1.The limitation of detection of single fluorescence quantitative PCR methods for the detection of Spotted Fever group rickettsia and Anaplasma was 1 copy/μL.The limitation of detection of single fluorescent quantitative PCR methods for the detection of Borrelia burgdorferi and Babesia was 10 copies/μL.The limitation of detection of the double fluorescence quantitative PCR method for the detection of Spotted Fever group rickettsiae and Anaplasma and the double fluorescence quantitative PCR method for the detection of Borrelia burgdorferi and Babesia was 10~2 copies/μL.2.From March to May 2021,324 samples were collected from Jingxi City,Guangxi Province,Xilin Gol League,Inner Mongolia Autonomous Region,Zhoushan City,Zhejiang Province and Mudanjiang City,Heilongjiang Province,including 276ticks and 55 mouses.In 269 tick samples,the positive samples of Spotted Fever group rickettsiae were 210,and the positive rate was 78.07%.The positive samples of Anaplasma were 2,and the positive rate was 0.74%.The positive samples of Borrelia burgdorferi were 12,and the positive rate was 4.46%.The positive samples of Babesia were 3,and the positive rate was 1.12%.In 55 mouse samples,the positive samples of Spotted Fever group rickettsiae were 10,and the positive rate was 18.18%.The other three pathogens were not detected in the rat samples.3.The target genes of positive samples were amplified by PCR,and tick-borne pathogen species identification and phylogenetic analysis were performed.Four tick-borne pathogens were detected in 324 collected samples,including four species of Spotted Fever group rickettsia(Rickettsia japonica,Rickettsia sp.JX,Rickettsia heilongjiangensis,Candidatus Rickettsia tarasevichiae),two species of Anaplasta(Anaplasma phagocytophilum,Anaplasma sp.SFQ),four species of Borrelia burgdorferi(Borrelia valaisiana,Borrelia sp.JX,Borreliella afzelii,Borrelia garinii)and one specie of Babesia(Babesia canis).One sample of Ixodes granulatus had the co-infection of Spotted Fever group rickettsia,Anaplasma and Borrelia burgdorferi.Two samples of Ixodes granulosus had the co-infection of Spotted Fever group rickettsia and Borrelia burgdorferi.One sample of Ixodes persulcatus had the co-infection of Spotted Fever group rickettsia and Anaplasma,and nine samples of Ixodes persulcatus had the co-infection of Spotted Fever group rickettsia and Borrelia burgdorferi.Conclusions1.In this study,four single fluorescence quantitative PCR detection methods were established,based on which two double fluorescence quantitative PCR detection techniques were established,covering nine species of Spotted Fever group rickettsia,seven species of Anaplasma,five species of Borrelia burgdorferi.and ten species of Babesia.The methods can be used for the detection of tick-borne pathogens in ticks and mouses,which had good practical value.2.Four kinds of tick-borne pathogens were identified from collected samples,including four species of Spotted Fever group rickettsia,two species of Anaplasma,four species of Borrelia burgdorferi and one species of Babesia.A novel species of Spotted Fever group rickettsia and a novel species of Borrelia burgdorferi were identified in collected samples from Jingxi city,Guangxi Province.A new species of Anaplasma was identified in samples collected from Mudanjiang city,Heilongjiang Province.3.Multiple tick-borne pathogens were found to be co-infected in ticks.Two or three tick-borne pathogens were found in samples collected from Jingxi city of Guangxi Province and Mudanjiang City of Heilongjiang Province,suggesting that the pathogen species in these areas were complex and diverse and worthy of further study. |
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