The Preparation And Preliminary Application Of Four Poultry Respiratory Disease Visual Detection Microarray

Avian influenza, Newcastle disease, Infectious bronchitis and Infectious laryngotracheitis are commonly observed respiratory disturbance diseases in poultry. These infections diseases with similar clinical symptoms and pathological changes, high fatality cause great economic loss for the poultry industry. Therefore, the detection and diagnosis for the prevention and control of these epidemic diseases are significance. In this study, we developed visual oligonucleotide microarray technology on the basis of traditional microarray coupled with asymmetric PCR to simultaneously detect AIV, NDV, IBV and ILTV. The biotin labeled asymmetric PCR products hybridized to the probe on the microarray were reacted with streptavidin-horseradish peroxidase conjugate (Strep-HRP) and 3,3'-Diaminobenzidine (DAB), resulting in brown spots that easily visualized by unaided eyes for qualitative analysis. This method is a new technology to detect poultry disease in the clinical application.1. Preparation of AIV nucleoprotein (NP) gene recombinant plasmid and the oligonucleotide probe designConserved DNA sequences of AIV-NP gene were obtained from Genbank, a pair specific primer was designed. The target fragment length is 272bp. cDNA was obtained from reverse transcription of RNA which was extracted from isolates H9. PCR products of the AIV-NP gene were collected and connected to PMD19-T, and then transformed to DH5a. AIV-NP recombinant plasmid had been constructed successfully by PCR amplification and identification of nucleicacid sequence; Meanwhile, NDV fusion (F) gene, IBV nucleocapsid (N) gene and ILTV thymidine kinase (TK) gene was successful recovered from the conserved bacteria.The positive template strand of AIV-NP, NDV-F, IBV-N and ILTV-TK fragments about 40 bp oligonucleotide probes was designed by Oligo7.0, which had 80?�3? Tm value. The probes have good specificity with the BLAST analysis2. Preparating and optimizating of the visual microarrayPreparation of visual microarray Visual microarray was prepared by microarray(?) Smart ArrayerTM 16 system. The oligonucleotide probe of a certain concentration were diluted with Baio(?) spotting buffer, and then spotted onto drying nylon membrane which was soaked in ultrapure water 30 min before use. The spotted probes were cross-linked to the surface of substrate by exposing to UV light and then preserved at 4? before useAsymmetric PCR amplification techniques This experiment used the asymmetric PCR technology by optimizing the concentration of bio-labeled forward primer and unlabeled reverse primer. The asymmetric PCR results were analyzed by high concentration agarose gel electrophoresis at low voltage.The single strand DNA products were could be clearly observed when the concentration of bio-labeled forward primer and unlabeled reverse primer were used at the ratio of 10:1.Optimization of microarray preparation and testing technology The conditions of microarray preparation and testing technology were optimized. The optimum conditions were shown as following:one times of sample point repeated number,25uM of oligonucleotide, one hours of hybridization time,50? of hybridization temperature,2000 times diluted of 1.0 mg/mL Streptavidin HRP Conjugate,5 minutes of DAB staining time.3. Effectivity of visual microarrayIn this study, the quality of AIV-NDV-IBV-ILTV visual microarray was evaluated by specificity, sensitivity and stability. The results show that the visual microarray has good specificity. Plasmids diluted to different concentrations hybridization was showed that the minimum is 1.0�10-5 ng/?l; The preserved visual microarrays at different time were tested randomly and the results showed that that retention period is up to180 days; Collected ninety-six tissue samples from Sichuan and Chongqing area were tested by microarray and RT-PCR/PCR.The result showed that two methods have accordance detection rates.This study developed the visual microarray, which can simultaneously diagnose AIV,NDV, IBV and ILTV,and can be apply to clinical diagnosis and provid a new method for diagnosis of poultry disease.