The Establishment Of Digital Droplet PCR Method For The Detection Of Viable Vibrio Vulnificus And Its Initial Application In Clinical Samples

Vibrio vulnificus(V.vulnificus)is one of the five common pathogenic Vibrio in human.The establishment of timely and credible detection methods has become an urgent need for V.vulnificus illness surveillance.Digital PCR(d PCR),as a representative of the third generation PCR,could calculate the copy number of the target gene in initial samples directly according to Poisson distribution.The method can achieve absolute quantification of DNA without standard curve.The ideal situation in the actual test is to analyze live cells only.Hence,the problem of distinguishing live bacteria from dead bacteria in the target samples deserves attention.Live bacterial assays based on d PCR are beneficial in providing reliable therapeutic basis for all stages of pathogenic bacterial infections in clinical practice.PurposeIn this study,an assay combining droplet digital PCR(dd PCR)with propidium monoazide(PMA)treatment was developed for detecting V.vulnificus.The actual detection performance and interference resistance of this method in clinical plasma samples were also evaluated.The purpose is to establish a new rapid and reliable assay for detecting V.vulnificus and to perform accurate quantification of viable V.vulnificus in clinical samples without the use of pre-calibrated V.vulnificus DNA reference samples.Methods and Results1.After the optimization of primer/probe,concentration of PCR reaction,and annealing temperature in amplification procedure,the first,the V.vulnificus vvh A-q PCR assay was established.Based on this q PCR method,a dd PCR assay was established after similar optimization steps.In the specificity validation experiments of the established method,the primers and probe did not have non-specific amplification reactions with other 20 closely related strains and clinical isolates of common pathogens.The detection rate of different isolates of the same species tested was 100%,so it has practical application capability.After split optimization testing,the optimal concentrations of primers and probe are 1.0μM and0.3μM,and the annealing temperature achieving the highest accuracy in the dd PCR assay is60°C.The established method showed good linearity between DNA concentrations of 0.44pg/μL-220 pg/μL with R~2=0.9960 in pure culture conditions,and it is capable of detecting V.vulnificus DNA samples as low as 0.258 pg/μL in 80 min under non-enrichment condition.2.The optimal strategy to distinguish live cells from dead cells is to treat samples with 100μM PMA for 15 min in the dark,and expose them to LED light with output wavelength of465 nm for 10 min.Moreover,The selected PMA treatment conditions at a working concentration of 100μM for the live bacteria assay study did not affect the amplification of live V.vulnificus DNA in the experiment.The ability to detect live V.vulnificus by the established method was also confirmed in this study.3.The agreement between PMA-dd PCR test results and pathogenic culture results is 90.8%.Moreover,the dd PCR method can detect weakly positive samples of V.vulnificus that cannot be isolated and detected by culture method.Comparative experiments on the detection ability of PMA-dd PCR and PMA-q PCR in plasma samples were performed.After the addition of PMA treatment,the R~2 of both q PCR and dd PCR assay did not show any significant decrease.It shows that PMA processing does not interfere with the accuracy of the assay in practical applications.The limit of detection(LOD)and the limit of quantitation(LOQ)in pure culture solutions of V.vulnificus are 29.33 CFU/m L and 53.64 CFU/m L in PMA-dd PCR.For artificial clinical sample tests in PMA-dd PCR,V.vulnificus could be detected at concentrations as low as 65.20 CFU/m L.However,the experimental results indicate that the newly established dd PCR method in this study is not suitable to be used for the direct detection of high concentration target samples and should be pre-experimented first.4.The feasibility of the heat lysis DNA extraction method combined with the dd PCR live bacteria detection method was also explored.The LOD of the PMA-dd PCR method combined with the heat lysis method of DNA extraction is 148.76 CFU/m L in plasma samples,which is significantly better than the detection performance of the q PCR method in the same situation.On the other hand,the above results demonstrate that the dd PCR assay is more resistant to the interference of amplification matrix impurities in practical applications,which provides a new idea for the future development of the rapid detection technology.ConclusionThe PMA-dd PCR assay established in this study provides a new research and development direction for detecting viable bacteria in clinical samples,which is important to improve public health security capacity and emergency response level of V.vulnificus infection.