The Preparation And Detecting Technique Of NDV-IBV-ILTV DNA Microarray

Newcastle disease(ND), Infectious bronchitis(IB) and Infectious laryngotrach-eitis(ILT) are mainly respiratory diseases of chickens in the poultry industry. The clinical symptoms of respiratory system disease, makes its detection becomes a problem. Rapidly and accurately diagnosis was of great significance.In this paper, NDV, IBV and ILTV were collected to be studied, the purpose of this study was to prepare a DNA microarray for simultaneously, rapidly detection of NDV, IBV and ILTV. The main completed work of this study was divided into three parts:1. The establishment of microarray probe plasmid bacteriaBy sequence alignment analysis of NDV, IBV and ILTV genome, two conservative genes were selected as gene probes, six gene fragments and locating gene probe fragment that amplified by RT-PCR/PCR were connected with pMD19-T Simple vectors, then transformed, the establishment of probe plasmid bacteria could be stored for a long time.2. The preparation of microarrayThis study was carried out to optimize the process, construction of the NDV, IBV and ILTV in the diagnosis of gene chip, and was verified by hybridization.This test determined the concentration of probe purification method and probe. Test using isopropanol precipitation method, three methods of the kit method, ethanol precipitation method for purification of IBV-M gene, the results show that the ethanol precipitation method IBV-M gene and purification of the highest concentration. And ultimately determine the use of ethanol purification gene probe precipitation method, purification, concentration is 590.0-1857.5ng/p,L, buffer concentration will sample probe concentra-tion dilution to 250ng/uL.This test determined and processing conditions of preparing chip system. Test at 60% moisture contact point sampling system in the amino substrate, the point after the completion of 80 ℃ for 2h, UV cross-linking 25min, 10sec 60-80℃ to 80℃ water, drying, drying centrifuge for 0.2% SDS, distilled water after cleaning, prepared chip.The experiment established a quality inspection method of the chip, chip hybridization detection evaluation of construction quality. Test after comparing Cy3-dCTP 50,100 and 150 μmol/L three concentration, ultimately determine the Cy3-dCTP concentration was 100 μmol/L. Through the comparison of adaptation and fixed location method, ultimately determine the signal point positioning using fixed circle positioning method. The chip after prehybridization, hybridization, washing, scanning, data extraction results. Determine the positive standard:SNRL^ 1.0, hybridization spot was visible, positive control and negative control group were normal3. The preliminary application of microarrayIn this experiment, the validity, specificity, sensitivity, stability and goodness of fit test verification of the chip. Test two multiplex PCR screened for the target nucleic acid marker, were two groups, multiplex PCR 1:NDV-F(1.2μmol/L), IBV-N(1.1μmol/L), ILTV-gB(1.0μmol/L); multiplex PCR 2:NDV-HN(1.2μmol/L), IBV-M(1.4μmol/L), ILTV-TK(1.1μmol/L). Test chip with full probe hybridization results showed the effectiveness of good chips; chip and NDV, IBV, ILTV, AIV, IBDV hybridization showed good specificity chip; the chip sensitivity test showed that the sensitivity was 20pg/μL; random extracted three different batches of microarray hybridization showed that chip high stability; application chip in Sichuan and Chongqing region of the twelve samples tested, the results coincide with the conventional PCR method was 100%, indicating that the chip anastomosis high.In a word, this study established a DNA microarray method for simultaneously, rapidly detection of NDV, IBV and ILTV. It provided a new method for rapidly diagnosis of three kinds of important avian respiratory diseases.