Establishment And Preliminary Application Of A Method System For Nucleic Acid Detection And Genotyping Of Human Brucella

Brucella are severely pathogenic and zoonotic infection with Brucella can cause brucellosis which can lead to disability or even death if not promptly treated.However,due to the wide range of brucellosis clinical manifestations,the general symptoms are atypical manifestations and are extremely easy to misdiagnose,and rapid diagnostic treatment can avoid more serious outcomes.Both pathogenicity and antibiotic susceptibility vary between different Brucella species,and typing of Brucella species facilitates clinically targeted drug use.Therefore rapid detection,and accurate typing of this Brucella is of great significance.However,all the existing detection methods and the""gold standard""for typing Brucella have disadvantages such as high danger,long detection period and poor sensitivity.In this study,a set of highly sensitive,specific and time-dependent assays were developed for the existing detection problems,including a one-step nested real-time PCR assay for Brucella hominis establishment and a method for Brucella species typing of human origin,and MLST typing of Brucella hominis.This method system can detect at three levels:subordinate,species,and type,which not only provides an efficient and highly specific rapid detection method for clinical diagnosis,but also provides guidance on clinical antibiotic use,and a molecular epidemiological study of Brucella in Yunnan region,which can clarify the local genotypic characteristics and establish improved guidance for subsequent detection methods.This research work mainly revolves around:1.The nested real-time PCR method has been developed in a one-step process for the detection of Brucella,and the reaction takes two steps and is completed in the same reaction tube;We optimized a peripheral annealing temperature of 68°C and an inner circumference of 58°C;The final concentration of peripheral primers was optimized to 400 nmol/L;The specificity and reproducibility of the results were evaluated as follows;Collected whole blood from 145 positive patients,whole blood from 55healthy people,and 50 Brucella strains were used as templates to compare the sensitivity difference between the new method established in this study and ordinary qPCR,serological culture,and it was found that the new method had 100 fold higher sensitivity than ordinary qPCR.2.The mqpcr method was developed to detect four Brucella species(sheep species,bovine species,swine species,canine species)infected with human Brucella spp.,and the method can detect four Brucella species simultaneously with good specificity and reproducibility as evaluated in detail;For sensitivity testing,a lower limit of detection of 10~3copies was achieved for porcine species/μL.The lower limit of detection reached 10~2 copies in sheep,dog,or cattle species/μL;The whole blood of 100positive patients was selected for preliminary application.The detection rate was 95%and all of them were sheep breeds.The Ct value was concentrated in the range of 31-35,indicating that the method was suitable for clinical detection.3.Based on the MLST genotyping method,nine housekeeping genes(gap,aro A,glk,dna K,gyr B,trp E,omp25,cob Q,and int-hyp)of collected clinical Brucella strains were genotyped and analyzed.Preliminary genotyping analysis was conducted on the molecular prevalence of Brucella in Kunming,Yunnan.The main epidemic strains in Kunming,Yunnan were consistent with the epidemic type in China,mainly ST8(98cases,accounting for 98%),At the same time,ST39 is also prevalent in Kunming area(2 cases,accounting for 2%);By comparing the genotype sequences of ST8 and ST39,a base difference was found,indicating that the MLST genotyping method is highly sensitive;A phylogenetic tree was established and it was found that ST39 was located in the branch of ST8,indicating that ST38 may be derived from ST8 mutations.This study successfully established a one-step nested real-time fluorescence quantitative PCR method for the detection of human Brucella.The method has been verified to be highly sensitive and suitable for detecting samples with low viral load,providing an efficient,highly specific,and safe detection method for clinical detection;Establishing a human Brucella species classification method based on quadruple fluorescence quantitative PCR,which has strong timeliness,high specificity,and safety,is of great significance for the development of clinical treatment strategies for Brucellosis;The application of MLST typing method to clarify the molecular status of the prevalence of brucellosis in Kunming,Yunnan Province can provide data support and theoretical guidance for establishing strategies for prevention and control of brucellosis and detection techniques in Kunming;The system can be used to detect,classify,and type brucellosis at three levels:subordinate,species,and type.It has important value and significance for public health prevention and control,and clinical diagnosis and treatment of brucellosis.