| Dengue infection, one of the most important mosquito-borne viral diseases of human, is now endemic in tropical and subtropical regions. The disease ranges from an influenza-like disease known as dengue fever to a severe, sometimes fatal disease known as dengue hemorrhagic fever/dengue shock syndrome. The incidence of dengue fever and dengue hemorrhagic fever/dengue shock syndrome has increased significantly over the last decades. Yearly, an estimated 80-100 million cases of dengue fever and about 250000-500000 cases of dengue hemorrhagic fever/dengue shock syndrome occur worldwide. Up to now, there is no effective measure to prevent and control dengue infections, and no vaccine is commercially available. As a result, early diagnosis is urgently needed, and providing early diagnosis is also another effective way to prevent and control dengue infection.DNA microarray technology is a powerful tool in genetic analysis of biological samples. DNA microarray also named as DNA chips, which will provide researchers with the opportunity not only to detect the dengue infection at early stage in a more sensitive, specific, speedy, efficient and automatic manner, but also to detect and genotype dengue virus gene simultaneously.DNA chips can be classified into two categories: cDNA chips and oligo chips. In this report, we develop both approaches, in a hope to develop reliable ways for early diagnosis of dengue infection.The probes of cDNA microarray were collected from dengue cDNA library. Firstly, a fragment covering the full-length cDNA of dengue virus serotype 2 was amplified by long-PCR; then the amplified products weredigested with Sau3A I, a base "A" was added to the two ends of all the Sau3A I digested fragments by Taq DNA polymerase. These fragments were purified and cloned into T vectors. After identification and amplification by PCR with the T-vector primers, we isolated 142 positive clones. The method used to prepare probes was nested-PCR. In this study, Cartesian PixSys 5500 arrayer was used to spot the amplified probes onto the aminosilane coated slides to manufacture the cDNA chip for dengue virus detection.Two methods were employed in steps of sample labeling with fluorescence, namely the reverse transcription and restriction display technology (RD), which was invented by Wenli Ma and Wenling Zheng, The statistical method of T test was used to compare and analyze the hybridization results. T test (paired samples) results showed that the signal intensities of the RD labeling were significantly stronger than those labeled by reverse transcription. So in the subsequent experiments, all of samples were labeled by restriction display technology. To improve the detection specificity and reduce the nonspecific hybridization, human genome DNA and E.coli DNA, were labeled and hybridized with this dengue chip at 42 52 and 62 respectively. High temperature hybridization system was explored at 62 Under this hybridization system, we can detect the dengue virus genes with relatively high specificity. Meanwhile, human genome DNA, E.coli DNA, hepatitis C virus cDNA and Japanese encephalitis virus cDNA were employed for nonspecific hybridization analysis, and finally 8 dengue specific probes were selected for dengue infection diagnosis, which can be used as the probes for a integrated microarray for multiple pathogens detection. In addition, nine dengue virus isolates were detected by the microarray, and all samples showed positive result. These positive results demonstrated that the dengue cDNA microarray is reliable for detection of dengue virus genes.Oligonucleotide microarray can be manufactured by in situ synthesis orby spotting large number of oligonucleotide probes directly onto the slide. As the first method has strict patent protection, we used the latter technique to fabricate the Oligonucleotide microarray. The gene sequences of dengue virus were aligned using BLAST software online to find specific regions suitable to design oligoprobes. The selected sequences were further analyzed with software Oligo6.0 t...
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