Development And Exploring Of TaqMan-based One-step Single-plex/Multiplex Real Time RT-PCR Assay For The Detection Of Ten Arboviruses

Objective: The purpose of this study is to establish a TaqMan multiplex real time RT-PCR system to detect the major arboviruses which may have spread risk and health significance in guizhou. Methods: 1.Ten arboviruses were included in the study after consulting extensive literature, then Full genomic nucleic sequences of 10 arboviruses were obtained through the GenBank database search. Using Bioedit software analysis to find the conserved regions of each arbovirus, primers and TaqMan probes were designed with the help of NCBI blast and DNAStar primer select, and the mutual impacts of primers and probes in multiple combination were also evaluated. 2.RNA templates of target genes were synthesized by in vitro transcription, then RNA standards were used to evaluate each single-plex real time RT-PCR assay, standard curves were established, sensitivities and reproducibility were calculated. Cross-reactivity of primer-probe sets for detection of all arboviruses were checked. 3.Ten primer-probe pairs were designed and appraised based on the related diseases or virus families. According to the grouping information, Several kinds of primer-probe pairs were put in the same system at the same time. Set temperature gradient or Different concentrations to improve the multiplex real-time fluorescence quantitative RT-PCR systems. 4. All viruses were used to select double real-time fluorescence quantitative RT-PCR system. 5. RNA standards were used to evaluate each double real-time fluorescence quantitative RT-PCR systems. Standard curves were established, sensitivities and reproducibility were calculated. Using the RNA standards and mosquito samples to test the specificity of the reaction systems. Results:1.DENV-1/DENV-2/DENV-3/DENV-4/JEV/WNV/TEBV/KFDV/MVEV/Y FV were selected as the main monitoring arboviruses. 2. RNA templates of target genes were synthesized by in vitro transcription. 3.The standard curves of DENV-1, DENV-2, DENV-3, DENV-4, JEV, WNV, TEBV, YEV, KFDV, MVEV were successfully established. The correlation coefficients were more than 0.99. The amplification efficiencies were between 87.20%~102.00%. The sensitivities were 101-102 copies/μL and variation coefficients were less than 3%. There was no significant non-specific amplification plots of the Cross-reactivity of primer-probe sets. 4. The standard curves of DENV-2/DENV-3、JEV/WNV were successfully established. The correlation coefficients were more than 0.99, The amplification efficiencies between 83.60%~98.06%. The sensitivities were 102~103 copies/μL and variation coefficients were less than 3%. There were no cross-reactions between DENV-1, DENV-2, DENV-3, DENV-4, JEV, WNV, TEBV, YEV, KFDV, MVEV, and mosquito samples which infected with DENV-2 were detected by the Double real-time fluorescence quantitative RT-PCR system of DENV-2/DENV-3. Conclusion:1.Single-plex real-time fluorescence quantitative RT-PCR systems of DENV-1, DENV-2, DENV-3, DENV-4, JEV, WNV, TEBV, YEV, KFDV, MVEV were established successfully. These systems have high sensitivity, good reproducibility and strong specificity. 2.Double real-time fluorescence quantitative RT-PCR systems of DENV-2/DENV-3 and JEV/WNV were established successfully.The sensitivities of these systems have comparatively high amplification efficiency, reproducibility and specificity. These systems need to be optimized in follow-up study to improve the sensitivity and amplification efficiency of the systems. 3.These systems based on double real-time fluorescence quantitative RT-PCR technology were used as a rapid and sensitive method in thedetection of the vitro transcribed RNA and mosquito samples.These systems provide a possible approach for the rapid and accurate multiplex differential diagnosis of arboviruses.