| Lyme borreliosis, a tick-bone zoonotic disease, is caused by Borrelia burgdorferi infection。 The diagnosis of Lyme borreliosis were based on the typical clinical manifestation and laboratory test. But in the case of no significant clinical symptoms, laboratory diagnosis is particularly important. But at present, there was still no serological diagnostic commercial standard kit for Lyme disease in China, and the rapid detection of Lyme borreliosis still needs to be improved.The purpose of our study was evaluating the diagnostic efficiency of the specific recombinant proteins in serodiagnosis of Lyme borreliosis, using statistics software to screen out the most appropriate antigens to support producting Chinese clinical ELISA detection kit with recombinant antigens for Lyme borreliosis. One the other hand, to develop a novel isothermal assay of recombinase polymerase amplification with lateral flow for Borrelia burgdorferi, which is more faster and convenient than many other tests.We cloned and expressed five specific recombinant antigens, Fla B.g (the central fragment of flagellin), OspC B.a, P39 B.g, P83-E4 B.g and VlsE B.a were cloned, expressed, and purified. Then their antigenicity were identified by western blot first. Then their antigenicities were evauated with ELISA test using the sera samples of Lyme borreliosis, syphilis and healthy controls. In addition, another recombinant antigen OspC B.a also used in ELISA test was provided by Laboratory of Lyme borreliosis. Results of ELISA tests were analysed with receiver operating characteristic curves (ROC) to evaluate the sensitivity and specificity of each antigen. The results of all recombinant antigens were analysed with logistic regression models to screen out the valuable antigens for serodiagnosis of Lyme borreliosis.A specific segment of recA gene in B. burgdorferi genome DNA was selected for Basic RPA. A lot of pairs of primers were designed for the recA amplification by Basic RPA method to screen out the most appropriate primers. Then a specific probe was designed to add in the RPA with lateral flow reaction. The products of RPA reaction could be visualized by using the lateral flow dipsticks. Gradient experiments were performed to find the best reaction temperature and the most appropriate time. The sensitivity and specificity of this method were evaluated. To evaluate the advantage of this novel method, twenty serum samples of the patients with Lyme disease were detected by the RPA with lateral flow assay, nested-PCR and real-time qPCR respectively, and the results were pairwise compared by chi-square test.The results demonstrated with the ROC curve of each antigen showed that Fla B.g had a good diagnostic value for IgG and IgM test (AUC:IgG=0.800 and IgM=0.849), but it also had a high positive rate of syphilis (IgG 89.9%, IgM 31.4%). The AUC of VlsE B.a IgG was just 0.736, but that protein had the lowest positive rate in syphilis (12.3%, P<0.05) than other proteins. In addition, OspC B.g had the highest diagnostic value in test of IgM (AUC=0.871). By means of analyzing the detection efficiency of the proteins the logistic regression model we obtained two antigens (OspC B.g and VlsE B.a) for IgG and two (OspC B.g and OspC B.a for IgM. The diagnostic value of the screened antigen in the model was improved. By means of the simulation of the interaction between antigens in the model we found that the interaction between Fla B.g and ospC B.a, Fla B.g and OspC B.g, OspC B.g and P39 B.g, effected the diagnostic value in the model. Using the three groups of the above hypothetical interactional proteins mixed respectively, the results of serological Ab tests conducted again with ELISA method showed that for the mixed antigens with OspC B.a and Fla B.g the serum diagnostic efficacy was not improved and the specificity was reduced.RPA with lateral flow assay based on recA gene was successfully established, and its specific amplification was only achieved from genomic DNA of Borrelia burgdorferi. There was no cross amplification in Escherichia coli and other non-Borrelia strains. The detection limit of this new assay was about 50fg of B. burgdorferi genomic DNA. Results of serum samples of the patients with Lyme borreliosis’detected by three tests showed that the detection rate of RPA with lateral flow assay was much higher than nested-PCR (P<0.05), and there was no significant difference with real-time PCR (P>0.05). Otherwise, the RPA assay was more effective than the two method for its short reaction time and convenience.In summary, we obtained 6 pure recombinant proteins of Borrelia burgdorferi. Three appropriate recombinant antigens, OspC B.g, OspC B.a and VlsE B.a, screened out by means of statistics analysis were useful for serodiagnosis of Lyme borreliosis. And that the effects of the interaction between OspC B.a and Fla B.g to the serological diagnosis of Lyme disease should be paid attention in future research. At the same time, we developed the recombinase polymerase amplification with lateral flow assay for the detection of B. Burgdorferi. The novel assay with high sensitivity and specificity can get a visible result without any additional equipment in 30 minutes. That is great suitable for rapid diagnosis of Lyme borreliosis in clinic point of care testing. |
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