A Study On Constructing Of NDV-IBV Microarray And Its Detection Method

Newcastle Disease(ND) and chicken Infectious Bronchitis(IB) are both serious infectious diseases. Microarray is a new kind of biotechnology developed recent years which can be used to detect diseases. In this study, NDV-IBV microarray detecting viruses was constructed which can detect NDV and IBV at the same time.1. The cloning of NDV-IBV microarray's target genesAnalysing the molecular biological characters and gene sequences of NDV and IBV from literatures and GenBank,5 pairs of NDV primers, 4 pairs of IBV primers, 2 pairs of AIV primers and 1 pair of IBDV primers were designed by DNA Club2.0 or Arraydesigner 2.0 softwares. The Ribonucleotide acids(RNAs) of NDV, IBV.AIV and IBDV were extracted with Viral RNA mini Kit and The target genes were amplified by RT-PCR Acquiring 5 NDV target genes, 3 IBV target genes, 2 AIV target genes , 1 IBDV target gene and 1 unknown gene(WZ) which were named as ND1, ND2, ND3, ND4, ND5, IB1, IB2, IB3, AI1, AI2, IBD1 and WZ .respectively.ND1,part of NDV HN gene, is 487bp ,ND2 , part of NDV NP gene, 135 bp, ND3 and ND4 , part of NDV L gene ,334bp and 540bp ,respectively,ND5,part of NDV F gene, 522 bp; IB1 and IB2 , part of IBV NP gene are 276bp and 1010bp ,respectively, IB3, part of IBV M gene, 315 bp; AI1, part of AIV M gene, 789 bp, AI2 , part of AIV NP gene, 522 bp; IBD1, part of IBD VP3 gene, 428 bp; WZ gene, 364 bp. 14 recombinant plasmids were cloned using pMD18-T Vector and identified by digestion of RE,PCR and sequence analysis, which were named as T/ND1 (F48E9) ,T/ND2 (Lasota), T/ND3 (Lasota), T/ND4 (F48E9), T/ND5 (Lasota), T/ND5 (F48E9) ,T/IB1 (SM) ,T/IB1 (M41) ,T/IB2 (SM) ,T/IB3 (M41) ,T/AI1, T/AI2, T/IBD1 and T/WZ. These recombinant plasmids provided the reliable materials for microarray .The clone of target gene is a key technique for constructing NDV-IBV detection microarray.2. Constructing NDV-IBV microarrayThe construction and detection method of microarray were developed in this study.(1) Preparation of microarray An aminated glass slide was used to make a microarray .Thetarget genes were amplified by PCR with templates extracted from 14 recombinantplasmids and purified by isopropanol. The located gene, length of 500bp was amplified with template of XDNA by PCR. The concentrations of products were from 161.88 to 1218.36ng/ul. Target genes were diluted with spotted buffers(baiao ) at certain concentrations,then spotted on aminated glass slides with Microarray Printing System(SpotArray ?4), the space of spots was 450um, the diameter of spot 220um, 19 spots of every gene in a row. The spotted slide was dryed at room temperature for 2hrs, hydrated for 10 Seconds, lighted with UV for 25 mins. and washed with 0.2%SDS soulution for 5 mins., A microarray was successfully made.(2) Preparation of probe Probes were labeled by PCR with fluorescence CY3.The concentrations of CY3-dCTP and dCTP were 4.0uM or 8.0uM and 100.0uM or 200.0 uM in PCR in a 50ul reaction system, respectively, and the other dNTPs concentrations were 200.0uM.The concentrations of the probes purified with DNA and RNA Purified Kit were from 39.09 to 145.76 ng/ul,(3) The test method and analysis of microarray The experiment developed the detect program of microarray and its main parameters. After denatured for l-2min. at 95-100C,a microarray was quenched in anhydrous alcohol , centrifugated and dried,then pre-hybridizated for 30-60min. at 44C.The probes was denatured at 95C for 5min. putting in ice-bath immediately.then diluted with cooled hybridization buffer.Adding 25-30ul hybridiziton solution to the microarray,hybridizated for 7-9h at certain temperature. Washed accordingly with washing solution l(2.0%SSC 0.1%SDS), 2(0.2%SSC 0.1%SDS)and 3(0.16%SSC 0.1%SDS) each for 3-5min., then centrifugated and dried.The microarray was scanned with Microarray Analysis System(ScanArray 4000), data were analysed and disposed with QuantArray?Ver.3.0 software and data-analysing software.(4) The construction of NDV-IBV...