High-throughput Sequencing And Multiplex Fluorescence Quantitative PCR Methods For Detection Of Tick-borne Pathogens

Ticks are widely distributed and feed on the blood of their vertebrate hosts.As an important vector of pathogens,ticks can transmit a variety of pathogens such as bacteria,viruses,protozoa,et al.Identifying tick-borne pathogens is important for assessing human and animal infection risk and it is usef?lfor tick-borne disease prevention and control.In this study,16S rDNA high-throughput sequencing method and PCR method of pathogen specific gene were used to study the characteristics of bacteria composition carried by the dominant tick species in different regions.We identified tick species collected in six areas by morphological characteristics and mitochondrial 16S rDNA identification.Species composition and abundance were analyzed using 16S rDNA high-throughput sequencing methods.In addition,we detected common tick-borne pathogens by PCR methods for specific genes.By morphological and gene identification,the ticks collected from Qi County of Shanxi Province were Haemaphysalis longicornis and Haemaphysalis japonica,from Guertu County of XUAR were Hyalomma asiaticum,from Wenquan City of XUAR were Dermacentor marginatus,from Yushu City of Qinghai Province were Dermacentor nuttalli,from Jingna County of Yunnan Province and from Qiongzhong City of Hainan Province were parasitic Boophilus micloplus.The results of 16Sr DNA high-throughput sequencing showed that Coxiella burnetii,Anaplasma phagocytophilum,Rickettsia and Ehrlichia were carried in ticks from Qi County,Shanxi Province.Ticks from Guertu County of XUAR carried Francisella microorganism.Coxiella and Rickettsia were carried in ticks from Wenquan City of XUAR.Ticks from Yushu,Qinghai Province carried C.burnetii,Rickettsia heilongjiangensis,A.phagocytophilum and Ehrlichia.C.burnetii,Rickettsia heilongjiangensis,A.phagocytophilum,Borrelia miyamotoi and Ehrlichia were carried in ticks from Jingna County,Yunnan province.Ehrlichia muris and C.burnetii were found in ticks from Qiongzhong,Hainan province.PCR methods showed that Borrelia burgdorferi,B.miyamotoi and spotted fever group rickettsia were detected in ticks from Qixian County of Shanxi Province and Wenquan City of XUAR.Ticks of Guertu County of XUAR carried B.miyamotoi.In Yushu City of Qinghai province and Qiongzhong City of Hainan province,B.burgdorferi,spotted fever group rickettsia and A.were found in ticks.B.burgdorferi,B.miyamotoi,spotted fever group rickettsia and A.phagocytophilum were found in ticks from Jingna County,Yunnan province.Through high-throughput sequencing analysis of 16S rDNA,a variety of pathogens can be detected simultaneously,including Coxiella,Rickettsia,Anaplasma,and Ehrlichia.However,some pathogens can only match to the taxonomic information of genus,and the genome species/type cannot be determined.This method can not detect B.burgdorferi and have poor efficiency in detecting B.miyamotoi in this study.PCR method can detect B.burgdorferi and B.miyamotoi.The combination of the two methods can complement each other,which is conducive to a more comprehensive understanding of the bacterial pathogens of ticks.Multiple real-time PCR method has good application for detecting tick-borne pathogens.It can provide high sensitivity and strong specificity to improve the efficiency of pathogen detection,and detects some microorganisms in the same PCR reaction tube to reduce cost and shorten testing time effectively.Borrelia burgdorferi and Borrelia miyamotoi are two pathogens transmitted by hard ticks and can cause diseases in human.Duplex real-time PCR method for simultaneous detection of those two pathogens was established on the basis of singleplex PCR.Primers and probes were designed according to the recA gene of B.burgdorferi and glpQ gene of B.miyamotoi.The sensitivity of duplex real-time PCR for recA and glpQ gene was 102 copies/?L,which was lower than singleplex PCR(101 copies/?L).In the dilution series ranging from 109-102,there was no significant difference between duplex and singleplex PCR in terms of CT value(P>0.05).Duplex real-time PCR also showed good specificity.Moreover,there was a good linear correlation between pathogen concentration and CT value.The change in concentration and the presence of other pathogens had no significant influence on the results.100 Hyalomma asiaticum and 100 Dermacentor marginatus were detected by duplex PCR,14 of which were B.burgdorferi positive with CP value between 33 and 38.In conclution,the duplex real-time PCR method established in this study can detect two pathogens simultaneously,and has good practicability in detecting tick samples.In this study,a triple real-time PCR method for simultaneous detection of B.burgdorferi,spotted fever group rickettsia and A.phagocytophilum was established,providing a method for the surveillance and detection of tick-borne pathogen.PCR primers and probes were designed according to the recA gene of B.burgdorferi,ompA gene of spotted fever group rickettsia and msp2 gene of A.phagocytophilum.The reaction conditions were optimized and the standard curves were drawn.Triple real-time PCR method was established on the basis of singleplex and duplex PCR.The sensitivity and specificity of the method were tested,and the influence of concentration difference on the reaction results was explored.The new method was used to detect tick samples to evaluate the practical application effect.The results showed that there was a good linear correlation between the concentration of each pathogen and CT value,and the detection effect of each channel was good.The sensitivity of the triple real-time PCR is lower than that of the singleplex and duplex PCR,but it still has good sensitivity(102 copies/?L)and specificity.Pathogen concentration influenced the detection results,and the high-concentration template had an inhibitory effect on the low-concentration template.Comparison of the effect of single and triple methods on spot fever detection showed that the positive rate of detection was different(chi-square test),and the detection results were moderate and highly consistent(Kappa consistency test).This triple method has good application for tick sample detection.