| Arbovirus, also called arthropod virus, is a group of virus which can propagate in susceptible blood-sucking insects, such as mosquitoes, ticks, moskits, midges, and so on. It could spread between human and animals through bites without making its host insects pathogenic. At present, the international community has discovered about 537 kinds of arboviruses and confirmed that more than 130 species in them could cause the human and animals viral diseases. The symptoms could be the skin rash, the hemorrhagic fever, and even virulent encephalitis. In the history of mankind, arbovirus has made thousands of people and animals illness or death, causing huge economic losses. Nowadays, arbovirus still makes serious infectious diseases in various countries in the worldwide scale. China is a large country and its geographical landscape is also complex, there are many arboviruses through the area. It's confirmed that the Type B encephalitis virus, the forest encephalitis virus, the Xinjiang hemorrhagic fever virus and Dengue fever virus exist and prevail through our country. In addition, as the international exchanges become more frequent, the possibility of offshore arbovirus disease spreading to China has increased significantly. Therefore, the early, rapid and effective examination of arbovirus infection is of great importance.Suspension array is new-born biochip technology, which can qualitative and quantitative analyze a number of indicators at the same time. It innovatively uses the cell size's microspheres as the carrier of detection probes, and suspended in liquid, providing the extremely good response environment for the biological response, which is advantageous to maintain the native conformation of the nucleic acid and protein. It has some obvious advantages, such as high-throughput, good repeatability, high sensitivity, the characteristics of flexibility, etc. In this study, the genome sequences of dengue virus, Japanese encephalitis virus, West Nile virus, forest encephalitis virus, yellow fever virus were collected to be studied. Then, universal primers and specific probes were designed, combining with suspension array, we establish a diagnostic assay which can identify and serotype those viruses to make a new approach for arbovirus's detection and identification.First of all, we collected these viruses' full genome sequences from Genebank and then use biological softwares (ClustalX, DNAMAN6.0) to analyze the similarity and homoloy of each virus. Based on this, we design a pair of universal primer in the NS5 region of flaviviruses. Furthermore, we design 8 type-specific probes (20 nt) during the RT-PCR amplification region (266 bp) to identify JEV, WNV, TBEV, YF and serotype DEN1~4 at the same time. Primers and probes with degeneration design could cover most of the virus isolated. The primer and probes were validated firstly on Internet for their specificity by BLAST. The probes were all 20 nt in length with TM values between 47~57℃. Then, the probes were covalently crosslinked onto different carboxylated microspheres. Hybridization and washing conditions were optimized. The hybridization results were analyzed by Luminex 200 system. The established assay was verified by detecting and serotyping several arboviruses. The repeatability, specificity and sensibility of the chip were also evaluated. Finally, simulated complex samples and clinical samples were detected by the array comparing with conditional RT-PCR methods to verify the method furthermore.Viruses raised by sensitive cells were amplified by universal primers. The results show that every virus has been amplified specificly and effectively. This confirmed that the primers were designed with good specificity and versatility. Through a series of pre-experiments, the optimum conditions were determined to be 52℃, 15~30 min. Under this condition, the established assay could differentiate arboviruses exactly without any obvious non-specific signals repeatedly and the coefficient of variation was less than 8%. Meanwhile, we use the established assay to detect several unrelated viruses, such as influenza virus (H5N1, H3N2), rabies virus, Chikungunya virus, Sindbis virus, Saint Louis encephalitis virus, and so on. The results confirmed that the assay has high specificity in detecting arbovirus. Using serial dilutions of JEV, WNV and YF cultures, this assay reproducibly determined the lowest detection limit to be approximately 1.4 PFU for JEV and YFV, while 14 PFU for WNV. It indicated that this method has high sensitivity and could be used for clinical testing.The assay has also accurately identified the simulated samples which were mixed with different types of Dengue virus. It indicated that the assay could be used to detect complicated samples. Compared with conditional RT-PCR, the test results of 55 clinical samples collected and stored by our lab, the accuracy of the assay was 98.2%, while the specificity of detection was 100%. Through Kappa analysis (consistency test), we obtainedχ2 =0 <χ2(1)0.05 =3.841, so P>0.05. It suggested that the two methods coincide with each other without any statistical differences.By combining RT-PCR, nucleic acid hybridization with suspension array, we establish a new method which could simultaneously detect JEV, WNV, TBEV, YF and serotype DEN1~4. Confirmed by a serious of experiments, this assay had good detection repeatibility, specificity and sensitivity. It could be used to examine not only sole virus samples but also complex ones. Through statistics analysis, this assay had good consistency with conditional RT-PCR, while it had enormous advantages in rapid, high-throughput screening.This research in domestic and foreign takes the lead in applying suspension array to arbovirus nucleic acid detection. Not only could examine the early viral infection, moreover, the entire experiment operating process is simple and fast. It may play a significant role in infectious disease's epidemiology investigation, the clinical diagnosis, disease control and prevention, anti-terrorism, and so on.
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