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Objective: To isolate and identify the pathogen of Dengue fever from Shenzhen city in 2005 - 2006, and to analyze the molecular characteristics of the isolated Dengue virus strain as well as to explore its possible origin., Methods: IgM and IgG of serum samples taken from 60 suspected Dengue fever patients were detected by ELISA and immunochromatography, and 9 specimens were positive. Nine samples from patients with early stage Dengue fever were used to isolate virus with C6/36 cell line and the positive cell cultures were identified by MGB fluorescent PCR. The type of isolated virus strain was determined by RT-semi-nested-PCR and fluorescent PCR. E gene of isolated virus strain was amplified by RT-PCR and sequenced. Homology and phylogenetic tree of E gene of Shenzhen Dengue virus with the strains isolated from other areas were constructed., Results: Of nine antibody-positive serum samples, one strain of Dengue virus was successfully isolated. The isolated virus strain was confirmed as Dengue virus type 2 and designated as DEN2-SZ0521. The homology of nucleotide sequence and the deduced amino acid sequence of E gene of SZ0521 with standard type 2 Dengue virus NGC strain was 94.2% and 98.2%, but the homology with standard Dengue virus 1, 3, 4 in the same fragment were 59.1%, 57.2%, 58.5% and 68.1%, 66.7%, 63.2%, respectively. The phylogenetic tree indicated that SZ0521 had the greatest similarity with the Malay0412a/Tw strain and they lied in the same branch of the phylogenetic tree. The corresponding homology of nucleotide sequence and amino acid sequence was 99.8% and 100%, respectively. The isolated Dengue virus type 2 belonged to genotype IV with Indonesia-76, Somalia-84 and Sri Lanka-90., Conclusion: Dengue virus was isolated from Shenzhen for the first time, and it was classified as type 2. It was confirmed that the type 2 Dengue virus may come from the epidemic area in Malaysia.

Objective: To develop the Taqman MGB real-time fluorescent PCR assay for rapid detection of Dengue viruses., Methods: Using Taqman MGB technique, a pair of universal primers and Taqman MGB probe were designed according to a highly reserved sequence of the 3'-noncoding region of dengue viruses type 1-4. Dengue virus strains were used as standard and Japanese encephalitis virus strains were used as control, the real-time PCR assay for specific and sensitive detection of the dengue viruses was established. While 8 serum specimens of ELISA positive were detected by the RT-PCR and fluorescent PCR., Results: The sensitivity of real-time PCR was 0.17pg/microl (cDNA)or 10(-5)TCID50. There was no cross-reaction with Japanese encephalitis virus. Of 8 specimens, 2 were positive by RT-PCR and 5 were positive by real-time PCR. The test could be completed in 4 hours., Conclusion: The Taqman MGB real-time PCR assay was fast, sensitive and specific. It could be applied to the quick early diagnosis of dengue viruses.