Establishment And Preliminary Application Of Double Fluorescent Quantitative PCR For Detection Of Ehrlichia And Borrelia Burgdorferi

With the improvement of economic level,people’s contact with the natural environment is gradually increasing.Tick borne infectious diseases such as Ehrlichiosis caused by Ehrlichia and Lyme disease caused by Borrelia burgdorferi have gradually attracted people’s attention.At present,there are mainly the following problems:(1)There is no PCR method for simultaneous detection of Ehrlichia and Borrelia burgdorferi,and the existing detection methods are limited.They are too complex to be suitable for rapid detection of clinical samples.(2)There is no systematic epidemiological investigation on Ehrlichia in China,and the distribution of Ehrlichia in China is not clear.It is of great significance to study the situation of Ehrlichia carried by ticks.(3)Since the first report of Lyme disease and its natural foci in 1983,there has been no systematic molecular epidemiological investigation on the pathogen of Borrelia burgdorferi in China.Tick is the main vector of Lyme disease.Systematic molecular epidemiological investigation on the situation of Borrelia burgdorferi carried by ticks is of guiding significance for the prevention and control of Lyme disease in China.In view of the above problems,this study established a dual fluorescent quantitative PCR diagnosis method for Ehrlichiosis and Lyme disease,and carried out preliminary application,in order to provide a simple and effective clinical diagnosis method;carried out molecular epidemiological investigation on Ehrlichia carried by ticks in some areas of China,in order to provide information for understanding the distribution of Ehrlichia;carried out molecular epidemiological investigation on Ehrlichia carried by ticks in some areas of China.Objective to provide a theoretical basis for the prevention and control of Lyme disease.The main contents are as follows:1.Establishment of a dual quantitative PCR and real-time PCR for detection of Ehrlichia and Borrelia burgdorferiIn this study,a double fluorescent quantitative PCR method was established to detect the Gro EL gene of Ehrlichia and Osp A gene of Borrelia burgdorferi.The specificity,sensitivity,stability and consistency with conventional PCR methods were studied.150 samples(25 groups)of Ixodes persulcatus from Heilongjiang Province,Jilin Province,Liaoning Province and Beijing were detected by this method.The results showed that this method could only specifically amplify the genes of Ehrlichia and Borrelia burgdorferi,but was negative for Leptospira,Rickettsia,Escherichia coli and Pseudomonas aeruginosa,which was consistent with the traditional PCR method.The detection limit of nucleic acid was 1×10~1copies/μL.The coefficients of variation of TM values in both intra-and inter batch repeated tests were less than 0.1%.The detection rates of Ehrlichia and Borrelia burgdorferi were 32%and 20%,respectively.The detection rates of traditional PCR were 24%and 16%,respectively.These results indicate that the method established in this study has good specificity,high sensitivity and strong repeatability.It can be used to detect both Ehrlichia and Borrelia burgdorferi at the same time,and can be used for molecular epidemiological investigation,and differential diagnosis.2.Molecular epidemiology of Ehrlichia carried by ticks in some areas of ChinaIn this study,1470 ticks were collected from cattle and dogs in Jiamusi and Yichun of Heilongjiang province,Siping of Jilin province,Dandong of Liaoning province and Beijing from 2017 to 2019.The ticks were divided into 354 groups and genomic DNA was extracted.The double fluorescent quantitative PCR method and the methods of Gro EL,DSB and 16S r RNA were used.Twenty groups of positive samples were identified by PCR and phylogenetic analysis,including Ehrlichia muris(10 strains),Ehrlichia ewingii(3 strains),Candida neoehrlichia mikurensis(3 strains),Anaplasma phagocytophilum(1 strain),Anaplasma bovis(1 strain),Ehrlichia ruminantium(1strain)and Ehrlichia Minasensis(1 strain).The total positive rate of samples was 5.64%,of which the positive rate of Heilongjiang Province was 5.23%(16/306);the positive rate of Ixodes persulcatus in Heilongjiang Province was 28.3%(15/53);the positive rate of Jilin Province was 9%(4/44);the positive rate of Liaoning Province was 0%(0/2);the positive rate of Beijing was 0%(0/2).3.Molecular epidemiology of Borrelia burgdorferi carried by ticks in some areas of ChinaIn this study,354 groups of tick genomic DNA were identified by nested PCR and ordinary PCR using the 5S-23S ribosomal RNA intergenic spacer,rec A and osp A gene primers of Borrelia burgdorferi and the double fluorescent quantitative PCR method was established by this project and the genetic evolution of the obtained sequences was analyzed.A total of 354 groups of samples were collected,and 11 groups of positive samples were found,all belonging to Borrelia garinii.The total positive rate was 3.1%(11/354);the positive rate in Heilongjiang Province was 3.27%(10/306);the positive rate in Heilongjiang Province was 18.86%(10/53);the positive rate in Jilin Province was 2.23%(1/44);the positive rate in Liaoning Province was 0%(0/2);the positive rate in Beijing city was 0%(0/2).