Primary Study Of Oligonuclitide Microarray For Detection Of Japanese Encephalitis Virus And Yellow Fever Virus

Japanese encephalitis is caused by the Japanese encephalitis virus (JEV) and transmitted by mosquito bites. It causes serious inflammation of the brain, which may lead to permanent brain damage, and has a high mortality rate. Worldwide, approximately 35,000-50,000 symptomatic cases develop per year. The disease is spread throughout mostly areas of Asia.Yellow fever is a viral disease that has caused large epidemics in tropical regions of Africa and South America. The disease is caused by the yellow fever virus (YFV). There are 200,000 estimated cases of yellow fever (with 30,000 deaths) per year.Both of JEV and YFV belong to the flavivirus group. The disease may be difficult to distinguish, especially during the early stages. Since there are no treatments available to stop or slow the progression, therefore, the early laboratory diagnosis of JEV/YFV is important to immediately plan proper patient management,to avoid misdiagnose or delay state of the illness.Traditionally laboratory diagnosis always relied on using immunological methods to detect the viral antigen. This method sometimes yields false-positive results because of cross-reactivity between JEV, YFV and other fiaviviruses. As an alternative, RT-PCR based assays have been developed for the detection JEV, YFV. However, the detection of RT-PCR product was unsatisfactory due to the interference caused by false-positive or false-negative result on occasion.Microarray represents the latest advance in molecular technology, providing unparalleled opportunities for multiplexed detection of nucleic acids. The technology offers tremendous potential for microbial community analysis, pathogen detection, and process monitoring in both basic and applied environmental science.To begin with, we used bioinformatics software such as BLAST, Oligo 6.0, DNAClub to select oligonucleotide probes of high specificity, identical length and similar melting temperature (Tm). After that they were aligned by BLAST to eliminate the sequences that were not specifics to their own genome. Seventeen and nineteen oligonucleotides were originally selected and synthesized for JEV and YFV, to printing into an oligo microarray for virus detection respectively.Extracted total RNAs of healthy person serum, the plasmids of dengue fever virus and hepatitis C virus(HCV) were used as negative controls. Plasmids, which were cloned in JEV gene fragments and YFV whole genome were used to evaluate the probe. Samples were labeled by restriction labeling method and were applied to the microarray for hybridization. The results were scanned by confocal scanner. The image was extracted by Genepix 6.0, data collected were compared to evaluate the probes and for the selection of candidate probes for further oligo microarray preparations.After the probe selection has been completed, the application of this microarray to a combined detection for JEV and YFV was studied. Gradating diluted the labeling sample to study the sensitivity of RD-PCR labeling method by Oligo microarray hybridization.Gene fragments of NSl,NS2a,NS2b,NS4a and NS4b were ligated with pMD18-T vector by A/T clone. Then they were labeled by traditional PCR and asymmetric PCR. The labeled samples were examined by the Oligo microarray, followed by washing and scanning under the same conditions. Comparing to the traditional PCR labeling method, the asymmetric PCR labeling method showed superior results with enhanced hybridization efficiency.We found that the different hybridization efficiency of each probe probability depended on the distance of the oligonucleotide position from the 3' end of the DNA target.Through experimental study, we reached the following conclusions:1. Comparison of different annealing temperature by using restriction display PCR, it has been demonstrated that we can get the better efficiency of amplification when we choose 55 °C 30s-58°C 30s-60°C 30s as annealing temperature.2. The sensitivity of the probes was evaluated.lt has been found that the sensitivity of 60mer oligonucleotide microarray is no less than 0.04ng/uL (0.15nmol/l) by diluting sample gradually.3. After experimental testing and excluding the non-specific oligos from the original microarray probes, 28 oligo probes were selected in the final format of oligo microarray to detect the JEV and YFV simultaneously .4. Target sites of the probe were tested. It is noteworthy that all oligonucleotide probes far from the 3' end of the target fragment showed a lower efficiency of hybridization than those probes located closer to the 3' end of the fragment.In summary, in the present study we used the combined restriction displayPCR-microarray approach for reliable and accurate detection of JEV and YFV. This method has great potential for application in clinical diagnosis.