Research And Application Of Diagnostic Assay For Poultry Respiratory Syndrome With Macroarray Techniques

A macroarray technique was developed by preparing highly conserved DNA fragments for the five poultry respiratory tract diseases using molecular cloning methods, and the DNA fragments were spotted onto NC filter to form macroarrays. RNA extracted from samples was reverse-transcripted then followed by PCR amplification and labled with biotin-16-dUTP. The labeled DNA and prepared DNA macroarray were subjected to specific hybridization and the hybridization results were scanned and analyzed with a scanner .The results shows that the macroarray technique could identify and distinguish the five poultry respiratory tract diseases.This research was divided into the following three parts:Part Ⅰ, Establishment of the macroarray techniques in identifying and distinguishing the five poultry respiratory tract diseases. Thedesigning and preparation, labling of target gene, hybridization and scanning of Macroarray, the efficiency appraise of Macroarray were included in this part.We amplified the special sequence and linked them into pMD-18T vector separately,then transformed them into Ecoli and identified them by PCR and detecting sequence.The result of gel electrophoresis shows special strips of positive clone at corresponding site. We compared the sequence with that in the Genbank of NCBI, the result shows that all kinds of detected virus are above 95% in homology .We distilled nucleic acid from positive plasmid and spotted them onto NC filter after purified, then we baked it for 2 hours at 80℃, finally we finished the preparation of Macroarray. We choosed different aperture NC filter from dissimilitude factory during the preparation. As a result, 0.65μm NC filter of ZheJiangHangyan was the best which couldn't distort and diffuse. The backgrounds was good than that of America. We attempted three kinds of labling methods in the course of detecting samples, such as mixing method, photosensitive biotin method and end labling method. And the result shows that biotin-16-dUTP mixing in sensitivity and specialty is better than photosensitive biotin and end labling method.At the same time, we optimized the hybridization time .The best time is 1hour at 42℃We can conclude that the supervising system and disease diagnosissystem is constant by the detection of 10 shares of negative samples and 20 shares of positive samples. There were evident hybridization signal at positive control site and corresponding site, while no signal at negative site.We diluted the NDV by 10 times aimed at the sensitivity detection. And we could detect hybridization signal at 10'10 times diluting, but the gel can only detect 10"6 time diluting. The result shows that the sensitivity of DNA chip is higher than PCR by 104 times.Nucleic acid were distilled from five kinds of pathogens separately, using reverse transcript PCR amplified them and labled by biotin, in mixing primer and single template .We can only find hybridization signal at corresponding site and no no-specialty hybridization site. Repeating test shows that, every site is good.Part II, Comparison among Macroarry technique, separation and culture, PCR method. We detected 10 positive samples of every kinds pathogen. The positive samples are identified by Oland biological engineer academy. The result shows that separation and culture method is most different from PCR and Macroarry technique (p<0.01), while the radio of the anastomose is above 90% in PCR and Macroarry technique, but the separation and culture is a bit low compared with themPart IE, tentative application of Macroarry technique. We diagnosis the doubtful samples with Macroarry technique and PCR method in Qingdao,Jining,Zhucheng et al.The result shows that, the radio of the anastomose is almost above 90%.At the same time, we compared 60 shares of swab samples and homogenate samples by Macroarry technique. As a result, there was the radio of the anastomose is above 80%.