Establishment Of Detection Methods And Co-infection Research For Brucellosis And Q Fever

Object:Bacterial load in clinical samples is relatively low and difficult to detect,particularly those that survive inside host cells and cause chronic infections,like brucella and Coxiella burnetii in this study.Low sensitivity usually results in a false negative result,which may cause a delay in diagnosis or a misdiagnosis.To detect brucella and Coxiella burnetii,we present a new strategy to improve the sensitivity of a nucleic acid assay by detecting the presence of a multi-copy gene and see if co-infection between them.Methods:(1)Brucella was used as a test case.The genome sequence of Brucella melitensis was screened and several high copy genes were successfully identified.Quantitative PCR to compare the detection efficiency,analysis of IS711 sequences.and then clinical sample detection.(2)Based on the multi copy gene IS1111 a,we designed primers and probe for Coxiella burnetii,In view of the pollution caused in the process of detection,we constructed a false positive plasmid using the fusion PCR method as a positive control,in this way to detect the DNA of ticks.(3)Detection of brucellosis and Q fever in Xinjiang region and the northeast region,to understand whether there is a co-infection between the two pathogens.Results:(1)Analysis of B.melitensis strain 16 M genome sequence,a total of 38 genes were found to have multiple copies.With IS711,BMEI1001,BMEI0775,BMEI0027 quantitative PCR of serial dilutions of16 M templates,IS711 showed better amplification efficiency.Clinical samples were detected with the four assays,target concentrations of IS711 and BMEI1001 were consistently higher than BMEI0775 and BMEI0027.Concentration of IS711 ranged from 1.92 to 5.92(95% CI 2.88-3.94),for BMEI1001,it ranged from 2.58 to 5.35(95% CI 2.91-3.94).From the 20 samples,IS711 was detected in 17,followed by BMEI1001 in 14,BMEI0775 in six,and BMEI0027 in five.(2)False positive control plasmid can replace true positive plasmid as control,greatly reducing the pollution,multi copy insertion sequence IS1111 a as a signature gene of Coxiella burnetii.178 samples of Baicheng total nucleic acid were detected by quantitative PCR,and 4 samples were Q fever positive.(3)Brucellosis and Q fever co-infection rate in Korla,Yili,Jimunai,Habahe,Atushi,respectively 6.94%?10.98%?4.85%?7.46%?11.76%,northeast China total co-infection rate was 0.Conclusion: Bacterial pathogens in clinical samples are difficult to detect because of their low concentration.Although more sensitive assays are being developed for detection of these pathogens,they are still limited by the low number of bacteria.In this study,we designed a new strategy that targets high copy number genes to improve sensitivity.The improved sensitivity is dependent on the copy number of a signature gene in bacteria.Since the genomes of nearly all known bacterial pathogens have been sequenced and annotated,it is possible to screen high copy target genes as signature sequences.Therefore,this strategy is universal and could be extended to other bacteria.