Establishment And Application Of Multiplex Subtyping Real-time Fluorescence QPCR Method For Detection Of Borrelia Burgdorferi Sensu Lato

Lyme disease,also called Lyme Borreliosis,is zoonotic diseases caused by infection of Borrelia burgdorferi and spread by ticks for the media.Borrelia burgdorferi is the major pathogen of Lyme disease,is spirochetes order,spirochetes family,Borralia genus in the taxonomy.Lyme disease gets its name because the first found in 1977 in the Old Lyme town of Connecticut in America.Lyme disease is a group of clinically heterogeneous diseases,early characterized with erythema chronicum migrans,then heart,joint,nervous system involved,severe cases are likely to die.Lyme disease is widely distributed in the world,spreading all over 70 countries in five continents,and the number of the infected is on the increase year by year with the highest in the United States.The existence of lyme disease is also confirmed in our country.Some provinces(cities) exist natural epidemic focus,which harm people’s health and economic development.WHO has classified the disease as a key object of prevention and treatment in 1992.Strengthening the etiologic detection of frontier ports becomes urgent with international economic and demographic communications growing,which provides possibility for the spread and transfer of pathogen.We must detect it earlier,prevent and control it earlier,to guarantee the safety and stability of border areas.At present,the main laboratory examination of lyme disease are hemogram analysis,pathogen isolation,serology and molecular biology methods.Pathogen separation is the gold standard whose positive rate is highest with the sampling of lesion surrounding skin,but is not the preferred method for it is time-consuming.Immunological techniques mainly include indirect immunofluorescent assay and enzyme-linked immunosorbent assay.It is quick and easy,but there are some false positives and negetives.Molecular biology methods mainly include conventional polymerase chain reaction and real-time fluorescence quatitative polymerase chain reaction.This research intends to establish a method of detecting lyme disease and genotyping of the strains for lyme disease quick screening in frontier ports with multiple real-time fluorescence quatitative polymerase chain reaction for its advantages of being rapid,highly sensitivitive,specific and quantitative.Objective:This study intends to research the monitoring and testing technology of lyme disease.We want to establish a detection method for genotyping of B.garinii,B.afzelii,B.burgdorferi sensu stricto with the multiplex real-time fluorescence quantitative PCR.Methods:The complete ospC genome sequences of representative strains of B.garinii,B.afzelii and B.burgdorferi sensu stricto were searched from Genbank and were aligned by Mega 5.0 software.Conventional PCR primers were designed by Primer Premier 5.0 software.PCR was done based on the nucleic acid of three standard strains model buy from ATCC,to construct standard plasmid by TA clone used as the standard plasmid template for fluorescence qPCR.The general primers and type specific Taqman probes were designed based on the sequences of standard plasmid.The report groups of three probes were Fam,Texas Red,Cy5 respectively.Single real-time fluorescence qPCR methods and multiplex subtyping real-time fluorescence qPCR method for B.garinii,B.afzelii and B.burgdorferi sensu stricto were established and its reaction system were optimized used standard plasmid as template.And its sensitivity and specificity were analyzed.Scientificity and availability of this method was verified by the detection of the genome of three standard strains by the developed method and primary application for the detection of ticks captured from Changbai port.Results:TaqMan real-time fluorescence qPCR methods for B.garinii,B.afzelii and B.burgdorferi sensu stricto had good sensitivity respectively. The lowest detecting limit of B.garinii was 40copies/μL, the lowest detecting limit of B.afzelii was 5.17copies/μL, the lowest detecting limit of B.burgdorferi sensu stricto was 3.78copies/μL. Multiplex real-time fluorescence qPCR for B.garinii,B.afzelii and B.burgdorferi sensu stricto had good sensitivity and specificity.The minimum of multiplex real-time fluorescence qPCR is B.garinii 40 copies/μL,B.afzelii 5.17 copies/μL,B.burgdorferi sensu stricto 38 copies/μL. There was no cross reaction by detecting Rickettsia and Francisella tularensis,this proved good specificity.The multiplex subtyping real-time fluorescence qPCR method this experiment established was applied for lyme disease detection of 72 ticks captured from Changbai port in May 2009.The results showed 9 Haemaphysalis longicornis were positive for B.garinii genotype,which was consistent with conventional PCR sequencing results,that proved the reliability of this method.Conclusion:Multiplex subtyping real-time fluorescence qPCR method for detection of Borrelia burgdorferi this reseach established can rapidly detect Borrelia burgdorferi sensu lato and subtyping,which is suitable for rapid detection of lyme disease at the frontier port.