| Dengue virus(DENV) is one of the mosquito-borne Arboviruses with the highest morbidity and the most harmful in the tropical and subtropical regions. The infection of DENV can cause dengue fever(DF), dengue haemorrhagic fever(DHF), and dengue shock syndrome(DSS) with high mortality. Although many kinds of DENV vaccines have been in the research process, only the tetravalent Dengvaxia produced by Sanofi Pasteur was first registered in Mexico in December 2015. Therein, the hot topic is to precisely and efficiently determine and evaluate the neutralizing effect of the antibodies stimulated by the vaccine, which is the bottle-neck in developing DENV vaccines.The traditional evaluation method of neutralizing antibody titer is the neutralization test(Neutralization Test, NT). Among those, plaque reduction neutralization assay(PRNT) is known as the "gold standard". However it is labor-intensive, time-consuming, and low-throughput. In contrast, the advantages of the micro-neutralization test(MN) are less reagents consumption and high-throughput with the same principle as PRNT. Currently,the quantitative reverse-transcription PCR(q RT-PCR) method based on Taq Man probe has been reported and widely used in the quantitative detection of the viral nucleic acid because of its high sensitivity, specificity and throughput. Previous studies suggested that the MN assay based on q RT-PCR could be used to evaluate the neutralizing antibody titer against virus, such as influenza virus, CMV and SV40. Therefore, in this study, a q RT-PCR assay based on one-step Taq Man probe has been developed and optimized to assess DENV2 RNA. Then, the q RT-PCR-MN assay with a series of optimization has also been established for assessing the neutralizing titer of Ab against DENV2.[Method and result]1 Development and optimization of one step Taq Man q RT-PCR assay for measuring DENV2DENV2 strain NGC was propagated in a large scale with C6/36 mosquito cells as MOI=0.1. The virus supernatant of the infected cells was harvested, concentrated, aliquoted and then stored at-80℃. The virus stocks were assessed by the plaque test, TCID50 and Immunofluorescence assay. The titers of the virus stocks were 2×106 PFU/m L and 2.53×105 TCID50/m L, respectively. Then, the primers and probe targeting the conserved region of NS5 gene of DENV2 were designed and synthesized. The NS5 gene was amplified by PCR and the RNA standard sample was produced via in vitro transcription. The result of DNA gel electrophoresis showed that the size of target gene was expected. Afterwards a 10-fold dilution series of the RNA standard sample was prepared and assessed by one-step Taq Man q RT-PCR to draw the standard curve. Then we addressed the best primer/probe ratio in the q RT-PCR reaction system through trial and error. The result suggested that LOD of q RT-PCR was 1.0×102-108 copy/μl. Comparing with the plaque test, 1 PFU was approximately equal to 7500 copies. It suggested that the sensitivity of the q RT-PCR assay was 0.013 PFU, which was about 75 times as PFU.2 Establishment of q RT-PCR-MN method for detection of neutralizing effect of DENV2 antibodyBased on the above studies, the dose of infection and time-dependent tendency has been determined.The results revealed that the optimal dose of infection was the most positive results could be got by q RT-PCR after BHK-21 cells was infected by 2000 TCID50 for 24 hours.. After BHK-21 cells was infected by the 2-fold serial dilution of virus for 24 hours, the amount of the virus in supernatants(without extraction) and the amount of the virus in cells(extraction) were positively correlated(the correlation coefficient r=0.998). Meantime, the titer in the supernatant of the infected cells was positively correlated with the amount of virus infectious dose(r=0.982). Herein, directly using the supernatant of infected cells as sample is feasible in q RT-PCR test. And variety of the virus infection at least two-fold dose can be distinguished. Therefore, q RT-PCR-MN assay was established to detect the neutralization titer for DENV2 in this study. The neutralizing titer of 35 serum samples was evaluated by q RT-PCR-MN and PRNT respectively. The results showed that their correlation coefficient r was equal to 0.928. The neutralizing titer of Mc Ab 4G2 tested by PRNT(IC50) was 0.17 μg/m L, while that tested by q RT-PCR-MN(IC50) was 0.10 μg/m L. The correlation coefficient r was equal to 0.944 between them. Comparing with the results of 35 serum samples tested by PRNT, we found that the sensitivity of q RT-PCR-MN assay was 100%(7/7, 95% CI: 0.59-1.00), the specificity was 96.43%(27/28, 95% CI: 0.82-1.00) and Kappa coefficient was 0.915. Furthermore, there was no statistically significant difference when the neutralization titers of 10 serum samples were detected by q RT-PCR-MN assay in three kinds of instruments simultaneously. It suggested that the novel established q RT-PCR-MN assay in the neutralization titers assessed has good comparability and suitable to mutual recognition among laboratories.[Conclusion]In this study, a micro-neutralization test method based on a one-step TaqMan q RT-PCR-based assay for evaluation the neutralizating titer of the antibodies against DENV2 was successfully established. Compared to the classical PRNT, the correlation, sensitivity, specificity and consistency of q RT-PCR-MN were better. The supernatant harvested from the DENV2 infected cells can be directly used for PCR, which is much simple, rapid and convenient for subsequent automatic detection. In addition, results got from different instruments in various labs were consistence using this method, which is beneficial for clinical application. It also showed that q RT-PCR-MN assay is not only suitable for rapid and effective detection and evaluation of neutralizing antibody titer stimulated by DENV vaccine, but also for the large-scale epidemiological studies when DENV outbreak. |
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