Genetic Diversity Of Rickettsiales Bacteria And Optimization Of Quantitative Real-time PCR Detection Method

Objective:The order Rickettsiales is a kind of vector-borne prokaryotic cell microorganism.Ticks play a critical role in the transmission of Rickettsiales.In this study,we detected Rickettsiales pathogens in tick samples collected in Jinzhai County,Anhui Province for two consecutive years by molecular biological methods,and recognized the diversity of Rickettsiales pathogens in this area,providing the scientific basis for the prevention and control of tick-borne rickettsial diseases.And to establish and optimize a Taq Man-probe quantitative real-time PCR(qPCR)assay for the detection of 7 important Rickettsiales pathogens and simultaneous identification of the infection types.Methods:In this study,880 ticks were collected from Jinzhai County,Lu’an City,Anhui Province,China in 2021-2022,and their species were identified and phylogenetically analyzed by morphological and molecular biology.The 16S r RNA gene(rrs)fragment of Rickettsiales in tick DNA was amplified using nested PCR and sequenced for detection and preliminary identification of Rickettsiales pathogens in ticks.For further identification,PCR amplification and sequencing of the species-specific citrate synthase(glt A)gene and heat shock protein(gro EL)gene were performed on 16S rRNA-positive samples for further genotyping and molecular characterization of the pathogenic bacteria.And based on the omp B gene of Rickettsia prowazekii,Rickettsia mooseri,and spotted fever group rickettsiae,the gro EL gene of Orientia tsutsugamushi,the 16S r RNA of Ehrlichia chaffeensis,the glt A gene of Anaplasma phagocytophilum and the com1 gene of Coxiella burnetii,we synthesized primers and Taq Man probes and optimized the reaction system and reaction process to same solution.The sensitivity,specificity,and reproducibility of this assay were evaluated and the assay was used for the detection of simulated and actual samples.Results:(1)From the 880 ticks collected at the sampling sites,a total of five species of ticks in three genera were identified by morphology and molecular biology:Haemaphysalis longicornis(62.39%,549/880),Rhipicephalus microplus(27.95%,246/880),Haemaphysalis hystricis(6.25%,55/880),Haemaphysalis flava(2.95%,26/880),and Amblyomma testudinarium(0.45%,4/880).Nested PCR detection of Rickettsiales pathogenic bacteria showed that a total of 13 pathogenic bacteria were detected,including 2 Rickettsia(Rickettsia japonica and Rickettsia sp.JZT14),5 Anaplasma(Anaplasma marginale,Anaplasma bovis,Anaplasma capra,Anaplasma platys,and Candidatus Anaplasma boleense)and 3 Ehrlichia(Ehrlichia minasensis,Ehrlichia sp.Yonaguni138,and Ehrlichia sp.strain WHBMXZ-43)and 3 newly identified Ehrlichia members,among which 3 detected Ehrlichia members had different genotypes from known Ehrlichia members,suggesting that they may be new genotypes.Rickettsia was detected in H.longicornis and H.flava,with positive rates of 1.64%(9/549)and46.15%(12/26),respectively;Anaplasma was detected in H.longicornis,R.microplus,and H.hystricis,with positive rates of 13.84%(76/549),25.20%(62/246),and 9.09%(5/55),respectively;Ehrlichia was detected in H.longicornis,R.microplus,and H.hystricis,with positive rates of 3.64%(20/549),27.24%(67/246),and 1.82%(1/55),respectively.Nucleotide homology and phylogenetic relationship analysis proved that these Rickettsiales were widespread in the survey area,and revealed their genetic relationships and diversity.(2)The cycle threshold(Ct)value of the standard curves of the 7 pathogens showed a good linear relationship with the number of DNA copies(R~2>0.99),the minimum detection limit was 10 copies/μL,showing good specificity.In the 96 tick nucleic acid extracts,Coxiella burnetii was detected in 1 sample and spotted fever group rickettsiae was detected in 3 samples.In the 80 blood samples from patients with undefined febrile illness,Orientia tsutsugamushi was detected in 1 sample and spotted fever group rickettsiae was detected in 2 samples,the results were consistent with nested PCR.Conclusion:(1)The results showed that a variety of Rickettsiales pathogens existed in ticks in this area,revealing a wide diversity of local Rickettsiales pathogens,some of the pathogens detected have high homology with known human pathogenic bacteria,indicating a potential risk of human infection and should be monitored by the local population.This study plays a complementary and perfect role in the diversity of local tick-borne Rickettsiales pathogens and provides the basis and ideas for the prevention and control of local tick-borne diseases.(2)Based on the established Taq Man-probe quantitative real-time PCR assay,the reaction system and reaction condition of the 7 important pathogens of Rickettsiales were optimized to the same solution.This method overcomes the shortcomings of using different reaction systems and reaction conditions for different pathogens,which can precisely identify the species of 7 important pathogens of Rickettsiales in clinical sample detections and is important for the infection type identification and laboratory detection time reduction to facilitate precise treatment of the patients.