| Dengue virus which causes one of the most prevalent arbovirus diseases can be transmitted to humans by Aedes aegypti and Aedes albopictus mosquitoes in the tropical and subtropical regions of the world. There are four serotypes of Dengue virus (DENV-1, DENV-2, DENV-3, DENV-4), all of which cause dengue fever, dengue hemorrhagic fever and dengue shock syndrome. Clinical diagnosis of Dengue virus infections is difficult due to the non-specific symptoms. Therefore, laboratory diagnosis is essential. Serology detections depend on the existence of antibody and are not suitable for rapid diagnosis. Molecular methods, especially for PCR, have been widely used in early diagnosis of dengue fever. However, new sequence variation can inhibit these specific methods. This study developed a rapid assay for detection of Dengue virus including new strains using 3'-endfragment anchored and tag primed amplification (NAT-PCR) in combination with Sanger sequencing. It also described another tag primed amplification of randomPCR followed cloning M13 PCR products and Sanger sequenging to detect the dual infection of dengure.For the assay integrating a NAT-PCR with a new Fast Sanger sequencing, the conserved 3'genome end was targeted for specific cDNA synthesis. Degenerated primers were used for second strand synthesis. On all primers a common artificial tag was added to the 5'end, facilitating tag primed amplification. Fast Sanger sequencing was started from the anchored 3'genome end. The resulting sequences were analyzed by BLAST。The assay is specific against Influenza A, Lassa and yellow fever virus with no amplification or sequences got from NAT-PCR respectively Sanger sequencing. The specific SYBR Green I QPCR assays for Dengue cultured strains were applied for quantifying the virual RNA and throughout the whole procedure of NAT-PCR optimization. This assay demonstrated a high sensitivity with a detection limit of 11-31 copies viral RNA/reaction for a two-step protocol and 100-500copies for a one-step protocol. All four serotypes of Dengue can be tested in one reaction in similar to multiplex Real-Time PCR with high efficiency. In this study, sixty samples were run in parallel each time, but for higher throughput a 96-well format can be used for most steps. This assay was evaluated with 25 clinical serum samples. The detection results were consistent with clinical diagnosis reports. The one-step RT-PCR gave better results for the samples tested compared with two-step, since more positive rate and longer sequences were obtained. A random PCR was developed to get uniform and nonbiased amplification for non-segment single-stranded RNA. In combination with TA cloning and Sanger sequencing for M13 PCR products, the detection limit was 100 copies viral RNA/reaction. Twenty-one positive colonies for DENV-1 and thirty-two for DENV-2 were identified from 96 colonies in dual infection of Dengue with 100copies for each type. This assay was not specific for Dengue, but used to detect all the non-segment sigle-stranded RNA in the serum samples. It was also evaluated with the same 25 clinical serum samples. The positive results were the same as clinical diagnosis reports, but one negative sample was found to be Hepatitis C virus infection.Due to the virus strain differences in virulence, in particular for the emergence of new variant strains may determine the rate and severity of dengue infection, the variation of dengue virus was paid much attention. Dengue virus including new strains can be detected with 5 hours using NAT-PCR in combination with Sanger sequencing. For the endemic regions, dual infections of Dengue two serotypes can be resolved and distinguished by the assay of random PCR in combination with TA cloning and Sanger sequencing.This assy also hase a capability for detection of other non-segment single-stranded RNA in serum samples. |
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