Establishment And Preliminary Study In Clinical Application Of Real-time PCR With TaqMan Probe For Rapid Detecting Brucella

Object：To establish a method of real-time PCR with TaqMan probe for rapid detecting Brucellaand then explore the preliminary study in clinical application with this method.We attemptedto provide the experimental evidence for the follow-up of the patients with brucellosisduring the treatment and after treatment.Method：We chose16S rDNA of Brucella highly conserved genes for designing the primers, andthen selected the genus-specific sites for designing the TaqMan fluorescence probes markedwith the FAM and BHQ1. According to the principles and techniques of real-time fluorescentquantitative PCR, we established the method of real-time PCR with TaqMan probe for rapiddetecting Brucella, and constructed the absolute standard substance for TaqMan probereal-time PCR. We also established the standard curve for absolute quantification throughoptimizing the real-time PCR reaction system. The established real-time PCR with TaqManprobe was applied to detect brucella in100blood samples from the50patients diagnosed asbrucellosis hospitalized in the infectious disease department of1st hospital of Jilin Universityfrom June in2012to August in2013, and60blood samples from the randomly selected30healthy volunteers at the same time by real-time PCR assay, and compared the results with theresults of standard agglutination tube test (SAT) and blood culture. Follow-up were carried in8cases diagnosed as brucellosis whose blood were collected to do the real-time fluorescentquantitative PCR to mornitor DNA load changes of Brucella at three time point, includingbefore the treatment,1month after treatment and the end of the treatment.Results:The established real-time PCR with TaqMan probe was100%specific for B.melitensisand there were no cross-reactivity with reference strains, showing an analytical sensitivity of 102copies/μL. The correlation coefficient and slope value of standard curve were0.996and-3.23respectively and the efficiency of real-time PCR with TaqMan probe was104.003%.Forty-nine of the fifty patients had a positive real-time PCR result. The sensitivity, specificity,positive, and negative predictive values of the real-time PCR with TaqMan probe methodwere calculated as96%,100%,100%,93.8%, respectively. The observed positive agreementbetween STA and real-time PCR with TaqMan probe was92%, of which, the Kappacoefficient is0.468(P=0.00). Eight of fifty patients were monitored the brucella DNA loadfrom initial diagnosis through post-therapy follow-up.We found that there was a significantlydecrease in bacterial DNA load around the eight patients one month later during the initiatingtherapy. Two cases still maintained low bacterial DNA loads after the end of therapy, althoughthe clinical symptoms disappeared.Conclusion:The detection result indicates that the established real-time PCR with TaqMan probe forrapid detecting Brucella method has the advantages of specificity, sensitivity and repeatabilityand it is quick and accurate. In our study the sensitivity, specificity, positive, and negativepredictive values of the real-time PCR with TaqMan probe method were higher than SAT andblood culture method. Real-time PCR with TaqMan probe method could be a useful tool inthe clinical diaognosis of brucellosis and epidemic monitor. Furthermore,the consistentpositive rate of RT-PCR/SAT was higher than the other two combinations,therefor theestablished RT-PCR could be used to detect brucella at the early stage of brucellosis with theculture cultivate or agglutination tests being negative. The real-time fluorescent quantitativePCR we set up could be used in the brucellosis patients’ follow-up after treatment and it couldprovide laboratory evidence for brucellosis clinical staging, the observation of the treatmenteffect, the monitor of palindromia.