The Establishment Of Fluorescent Microarray And Visualized Microarray For Three Types Of Viral Hemorrhagic Fevers

Viral hemorrhagic fevers refer to a group of infectious diseases caused by four families of RNA viruses. All of these illnesses are characterized by^fever and bleeding. At present, there is no effective vaccine and therapeutic drugs for most viral hemorrhagic fevers.Crimean-Congo hemorrhagic fever is a tick-borne viral disease caused by Congo Crimea hemorrhagic fever virus (CCHFV). There are reports of infection cases from more than 30 countries in Africa, Asia, South-Europe, and the Middle East. Rift Valley fever is a zoonotic disease caused by Rift Valley fever virus (RVFV). The disease is epidemic in the African, the Middle East and the Yemen area, can harm personal safety and the development of animal husbandry. The Lassa virus (LAV) causes Lassa fever, a viral hemorrhagic fever that is endemic in West Africa. Because the disease is highly contagious, some European and American countries have also appeared infection case. At present, techniques for the diagnosis viral hemorrhagic fever include virus isolation, detection of specific antibody responses, and detection of the viral nucleic acids. Viral isolation is typically time-consuming and difficult and should not be introduced into a non-endemic area. The serological detection method may produce cross reaction and should pay attention to the sample collection time. Gene chip technology has the advantage of high throughput, microscale and highly automatic. So it has great potential in pathogen detection. Nevertheless, the requirements for special conditions and equipment restrict its wide application. In recent years, gold nanoparticles as oligonucleotide labels instead of fluorescent marker have been used increasingly in microarray-based DNA detection owing to high stability, pollution-free and inexpensive attributes.The aim of this study was to establish a method to detect CCHFV, RVFV and LAV using fluorescent gene chip and visualized gene chip. Three pairs of primers and six oligonucleotide probes were designed in accordance with the CCHFV, RVFV S gene and partial GPC gene and NP gene of LAV. The reverse primer were modified with fluorescent or biotin. The conserved sequences from the three types of viruses were cloned into pMD18T-simple to gain recombinant plasmid pMD-CCHFV, pMD-RVFV, and pMD-LAV. Using multiple PCR amplified and labeled purpose fragments. In vitro transcription reactions was used to synthesize RNA as detection template. The hybridization results of fluorescence gene chip were obtained by fluorescence microscopy. The amplification product which were labeled with biotin hybridized with probes on the gene chip. The gold nanoparticles labeled streptavidin was incubated, then formed the biotin-streptavidin-gold nanoparticle compounds. Finally, the signal was amplified again by silver enhancement staining. Finally, two kinds of gene chips were evaluated by specificity test, sensitivity test and repeatability test.This study has established fluorescent gene chip and visualized gene chip for the detection of three types of hemorrhagic fever viruses with high sensitivity and specificity. The sensitivity of fluorescent gene chip for the amplification product of pMD-CCHFV, pMD-RVFV, and pMD-LAV are 2.1×10-4 ng/ml,2.82×10-2 ng/ml and 1.73×10-4 ng/ml, respectively. The sensitivity of visualized gene chip for the amplification product of pMD-CCHFV, pMD-RVFV, and pMD-LAV are 2.1 × 10-3 pg/ml,2.82 × 10-1 pg/ml and 1.73 × 10-2 pg/ml, respectively. Compared with fluorescent gene chip, the sensitiveness of visualized gene chip improves 100 times (CCHFV),100 times (RVFV) and 10 times (LAV). In conclusion, the two kinds of gene chip with high sensitivity and specificity can be used for rapid detection of the three kinds of hemorrhagic fever virus.