The Establishment And Elementary Application Of The Visualized Microarrays For CHIKV, SINV And JEV

Chikungunya Virus (CHIKV) and Sindbis Virus (SINV) are single strand RNA virusesbelonging to Alphavirus, which spread through mosquitos and insects. They are distributedextensively around the world and have the characteristics of rapid pathogenicity andspreading. The prevalent of CHIKV and SINV make a threaten tothe global public safety andhealth. At present, there are no effective drugs for the diseases caused by both viruses.Therefore, early and efficient diagnosis of the viruses is the significant measures forprevention and control as well as restriction of the spread of the viruses.Japanese encephalitis virus (JEV) is RNA virus which can cause the serious viralencephalitis disease. The virus is transmitted between animal and human by means of culexmosquito and is prevalent in the eastern and southern area of Asia and the Pacific Ocean. Atpresent, vaccine for JE has been used to control the disease, but no cure drug for it. Therefore,early and effective diagnosis for JEV is extremely significant.Gene chip technology has the advantage of high specificity, strong sensitivity, high-flux,that provide a detective method for the clinical and environmental monitoring. Currently,there are a variety of gene chip technologies, marked with fluorescence, isotope, enzyme.However, they have many problems in low sensitivity, too complex operation, the need forspecial devices and conditions. Nanogold-modification method has the superiority of100times of more sensitivity in comparison with fluorescence gene chip, rapid and convenientdetection, with no need for special equipments and directly visible result in our eyes. Thisstudy apply the technology of nanogold-modification to the preparation of gene chip for theestablishment of visualized gene chip to the rapid, exact, cheap, handy detection of CHIKV,SINV and JEV. We respectively discuss two aspects of the assay: the establishment andcomparison of visualized gene chip for the detection of CHIKV and SINV; the praperationof visualized genotyping gene chip for detecting JEV. The main content is as following:The establishment and comparison of visualized gene chip for the detection ofCHIKV and SINVOur lab has already established the detection method of fluorescence gene chip forCHIKV and SINV. Based on this, we reestablish the two detective methods for CHIKV andSINV: fluorescent gene chip and visualized gene chip. We use the CHIKV and SINV E genesequence as template to redesign and modificate the probes and primers, amino-modification for probes and fluorescence/biotin-modification for primers; utilize the doting machine to putthe probes on the glass slide, and make the probes covalently bond to the glass slide; at thesame time, take the plasimid of CHIKV and SINV as template to amplify the targetingsequence by PCR method; degenerate the targeting sequence and hybridizate to the probes;wash out. For the fluorescence gene chip, the result can be got by special scanner. While, forthe visualized gene chip, the result can be eyed through marking nanogold on the targetingsequence taking the advantage of the speciality of high-affinity between biotin andstreptavidin and enhancing the signal by gathering sliver. The E genetic sequences of CHIKVand SINV correctly identified by enzyme-cutting are took as tempalte and reverselytranscripted into RNA modeled the viral CHIKV and SINV RNA. We respectively complexthe CHIKV and SINV RNA with extracted RNA of PRRSV, JEV-III, AIV and then obtain thecDNA of viral mixture by reverse transcription; qualificate the existing of viral cDNA byPCR and electrophoresis; amplify the viral cDNA using the primers for CHIKV and SINV;hybridize the amplification products to the probes on the glass slice; at last, undertake thespecific test with visualized gene chip. Meanwhile, the sensitive and repetitive test has beendone. The result show that the sensitivity of CHIKV and SINV measured respectively byvisualized gene chip and fluorescent gene chip are2.4×10-6ng/mL,2.0×10-8ng/mL and2.4×10-4ng/mL,2.0×10-5ng/mL. Comparison the gene chip methods to RT-PCR areapparently distinctive. The sensitivity of visualized gene chip is100times higher than that offluorescent gene chip. The specificity and repetivity of visualized gene chip is well.The praperation of visualized genotyping gene chip for the detection of JEVOur lab has already established the genotyping fluorescent gene chip for JEV. Based onthis, we set up the genotyping visualized gene chip for JEV. We dote the amino-modifiedprobes on the glass slides and get them imobilizated; biotin-modificate the existing primers inthe5’-end; amplify the targeting sequence using the palsmids of JEV-I and JEV-III whichcontain the PrM and E gene sequence as template; hybridizate the denatured targetingsequeces to the probes; make use of the high affinity of biotin and streptavidin to mark thenanogold on the targeting sequence; enhance the result of hybridization with sliver to achievevisualization. The sensitive, specific and repetitive tests get the result that the sensitivity ofgenotyping gene chip for JEV-I and JEV-III are8.1×105copy/mL and7.9×105copy/mL, andthe specificity and repetivity is well.