Treponema Pallidum Nucleic Acid Detection And The Study Of Droplet Digital Polymerase Chain Reaction Method For Absolute Quantification Of Treponema Pallidum

Part 1 Amplification of Treponema pallidum DNA from Whole Blood of Patients with Syphilis by Nested PCR AssayObjectives To investigate the contribution of nested polymerase-chain-reaction assay for the detection of Treponema pallidum DNA from blood of patients with different stages of syphilis.Methods Nested polymerase-chain-reaction assay(TP0105-Tp-PCR and TP0574-Tp-PCR)was used to detect Treponema pallidium in whole blood samples collected from 192 patients with syphilis(37 patients with primary syphilis,92 with secondary syphilis and 63 with latent syphilis).Results The detection rate in patients with primary(48.6%)and secondary syphilis(62%)were higher than those with latent syphilis(7.9%)(p<0.001).There were no significant different between the two target genes(TP0105 and TP0574)(p=0.69).The kappa coefficient was 0.714.Conclusion TP DNA could be amplified from whole blood samples from different stages of patients with syphilis,which indicated that spirocheta might be throughout the course of syphilis infection,but mainly in early syphilis such as primary syphilis and secondary syphilis.Nested PCR technique in which played an important role in the diagnosis of early syphilis,can be used in clinic to supplement the dark field microscopy and serological detection of supplementary diagnostic criteria.Part 2 Development and Application of a new Sensitive Assay Based on Droplet Digital PCR for the Quantification of Treponema pallidum Nucleic acidDroplet Digital PCR(dd PCR)now represents an affordable and powerful single molecule counting strategy to detect minute amounts of genetic material.There was no correlative literature of using dd PCR to accomplish Treponema pallidum nucleic acid detection.This study established a absolute quantitative PCR platform for Treponema pallidum nucleic acid detection,which was evaluated from the aspects of specificity,sensitivity,credibility and reproducibility.Put this platform to the detection of nucleic acid in peripheral blood of TP infected rabbit model to determine the feasibility of the technique.In this study,specific probes targeting TP0105 and TP0574 were designed,and the target gene fragments in healthy,non-syphilis,normal rabbits and mice were detected by dd PCR technique.The limit of blank of this technique was determined as 3.2copies(TP0105)and 1.4 copies(TP0574).Cut-off values were determined for subsequent detection of clinical samples.The nucleic acid quantification of each dilution gradient plasmid was determined by dd PCR technique to determine the high sensitivity of this technique,and the limit of detection(LOD)was regard as single copy.The correlation between the actual copy number detected by dd PCR and the calculated copy number was found to be highly correlated,indicating that the detection result of dd PCR was credible.In order to evaluate the reproducibility of dd PCR,we performed three replicates of the testicular suspensions of each dilution gradient.The results of the three copies were analyzed.It was found that the RSD values of each dilution gradient were within the acceptable range,indicating that dd PCR has good repeatability.In the comparative study of dd PCR and q PCR,we found that dd PCR had a lower detection limit than q PCR,higher sensitivity,and dd PCR had better reproducibility than q PCR,especially in cases where the target gene was at a low copy.In this study,dd PCR was used to dynamically monitor the infection of TP in rabbit.It was found that the appearance of nucleic acid in peripheral blood of infected rabbit was earlier than serological level,and the feasibility of dd PCR was confirmed.The absolute quantitative study of nucleic acid laying the foundation for the guidance of early childhood syphilis,fetal syphilis,neurosyphilis and other early diagnosis which is of great significance.