Establishment Of Real-time Quantitative PCR For Brucella And Research On Diagnosis And Efficacy Monitoring Of Brucellosis

Objective:Establish a real-time quantitative PCR method for the detection of Brucella,and test its role in the diagnosis of brucellosis,the effect of treatment,and the role of follow-up for relapse after treatment,which can provide a reliable basis for the early diagnosis of brucellosis and guide the antibacterial treatment to control the spread of brucellosis and reduce the disability and relapse rate of the disease.Materials and Methods:1.Research methods:?1?Preparing the standard samples from blood culture specimens of Brucella-positive patients,and then perform PCR and multiple PCR amplification on the standards and identify them by 1%agarose electrophoresis;?2?Everyone retained peripheral EDTA anticoagulated blood,extracted DNA within 24hours,and stored at-40?;?3?One-step instrument's SYBR Green mode was used to perform real-time quantitative PCR?qPCR?detection on all DNA specimens;?4?Brucella DNA load?Bru-DNA?and its logarithmic value?log10?Bru-DNA??of all specimens were calculated according to the standard curve and Ct value.2.Research objects and groups:All patients and healthy people were from the First Hospital of Jilin University between January 1,2018 and January 15,2020,with126 cases in the brucellosis group and 94 cases in the control group?including 46 other infectious diseases patients,48 healthy people?.126 patients in the brucellosis group were followed up for 3 weeks during treatment and 1,3,and 6 months after treatment,52 patients completed follow-up.According to the efficacy of 3 weeks during treatment,they were divided into 47 valid group and 5 invalid group;44 cases in non-relapsed group and 8 cases in relapsed group according to whether relapse occurred during the follow-up period after treatment.3.Statistical analysis and drawing method:SPSS 26.0 and R Studio 3.5.3 software were used for statistical analysis.Measurement data were first tested for normal distribution and homogeneity of variance.Data that conformed to the normal distribution were expressed as mean±standard deviation.Comparisons between different groups were performed using independent sample t-tests.Comparisons between same group at different times were performed using paired sample t-tests.Normally distributed data are expressed as median and quartile M(P₂₅,P₇₅).Mann-Whitney U test is used for comparison between different groups and paired Wilcoxon test is used for comparison at different times in the same group.For fewer?n?5?comparisons,a permutation test is used.Categorical data are expressed by a number of cases or percentages and?² test is used.P<0.05 was considered statistically significant.The amplification curve,standard curve and melt curve are from the software of the qPCR device.The ROC curve was drawn using SPSS26.0,the study grouping and design figure was drawn using viso2019,and the rest were drawn using R Studio 3.5.3.Results:1.The establishment of Brucella qPCR test method:The obtained standard was amplified by PCR and multiplex PCR,and identified by 1%agarose electrophoresis.The PCR product showed a fluorescent band at 904bp,the multiplex PCR product showed fluorescent bands at 208bp and 733bp.The electrophoresis band was consistent with Brucella melitensis.The standard curve R² of qPCR experiments is 0.992?0.984,0.998?,and all of them are greater than 0.980;the amplification efficiency is 93.0%?87.2%,99.0%?;the melt curve shows that the dissolution peak is about 75.5?,and the main peak is single.2.The role of Brucella qPCR in the diagnosis of brucellosis:The log10?Bru-DNA?of the brucellosis group was 4.26?3.83,4.91?,and the log10?Bru-DNA?of the control group was 2.64?2.34,3.03?.There was a statistical difference between two groups?z=-12.382,P<0.001?.In the two subgroups of the control group,the log10?Bru-DNA?of other infectious disease subgroup was 2.66?2.57,3.10?,and the log10?Bru-DNA?of the healthy subgroup was 2.64?2.24,3.03?.There was no statistical difference between two subgroups?z=-0.537,P=0.575?.The AUC of ROC reaches 0.988?95%CI:0.978-0.999?.The optimal cut-off value for Bru-DNA is 2.94E+03copies/ml,and its sensitivity and specificity were 96.8%and 95.7%,respectively.3.The role of Brucella qPCR in monitoring the treatment of brucellosis:Comparing the log10?Bru-DNA?between before treatment and 3 weeks during treatment in the valid group and invalid group,the 47 patients in the valid group was4.26?3.94,4.90?before treatment and 3.53?2.85,3.93?3 weeks during the treatment.There was a statistical difference?z=-5.736,P<0.001?.The five patients in the invalid group were 3.6284?4.3766?4.9736?5.1038?7.6274 before treatment,and 3.9435?4.6893?5.3444?4.3404?4.6776 3 weeks during treatment,and there was no statistical difference?z=1.214,P=0.156?.4.The role of Brucella qPCR method monitoring of relapse in the follow-up period after treatment for brucellosis:Comparing the log10?Bru-DNA?of the different time in the non-relapse group and relapse group.Before retreatment,the non-relapsed group was 4.25?3.90,4.89?and the relapsed group was 4.74?4.07,5.82?.There was no statistical difference between two groups?z=-1.268,P=0.214?.For 3 weeks during treatment,the non-relapsed group was 3.53?2.86,3.95?,and the relapsed group was4.18?3.45,4.68?.There was no statistical difference between two groups?z=-1.801,P=0.073?.In the follow-up period after treatment,the non-relapsed group was2.89?2.65,3.15?,and the relapsed group was 5.66?3.93,6.09?.There was statistically different?z=-3.779,P<0.001?.The results of log10?Bru-DNA?before treatment,3 weeks during treatment and the follow-up period after treatment of 52 follow-up patients on the risk of brucellosis relapse suggest that there was no statistical significance before treatment and 3 weeks during treatment.The follow-up period after treatment was statistically significant,and its OR was 23.557?95%CI:1.820,304.874?.ROC curve of 3 indicators?log10?Bru-DNA?for follow-up period,the change of log10?Bru-DNA?between follow-up period and before treatment,and the change of log10?Bru-DNA?between follow-up period and 3 weeks during treatment?in predicting the relapse of brucellosis indicate that the AUC of follow-up period was the highest,and the optimal cutoff value of Bru-DNA was 5.58E+03copies/ml.At this time,the sensitivity was 87.5%and the specificity was95.5%for predicting the relapse of the disease.Conclusion:1.The qPCR method for test Brucella DNA established by us has good stability,single product,good repeatability and meets the requirements of experimental design.2.The best cut-off value to distinguish brucellosis from non-brucellosis by detecting the DNA load of Brucella by qPCR is 2.94E+03copies/ml,and its sensitivity and specificity were 96.8%and 95.7%,respectively,the area under the ROC curve was0.988?95%CI:0.978-0.999?.3.The Brucella qPCR method was used to monitor the Bru-DNA of the patients before treatment and 3 weeks during treatment,and through their changes,the treatment effect can be better judged.4.The results of qPCR monitoring of 52 brucellosis patients suggest that the relapse of brucellosis is related to the DNA load of Brucella in peripheral blood after treatment,the best cutoff value of Bru-DNA for predicting the relapse of brucellosis patients is 5.58E+03copies/ml,sensitivity is 87.5%,and specificity is 95.5%.The OR value of Brucella DNA logarithm during the follow-up period after treatment was23.557?95%CI:1.820,304.874?,indicating that monitoring the Brucella DNA load of brucellosis patients after treatment by qPCR has a good effective in predicting the relapse of brucellosis.