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| Construction of Gene Chip for Detecting NDV-IBV-ILTV of Chicken (Gallus gallus) with Asymmetric PCR |
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Abstract Gene chip is a new biological technology based on the principle of nucleic acid hybridization, which shows important applications in the field of life science research. A method of simultaneously detecting Infectious laryngotracheitis virus (ILTV), Newcastle disease virus(NDV) and Infectious bronchitis virus (IBV) using DNA microarray and asymmetric PCR methods was developed. Primers based on specific conservative DNA fragments of the recombinant plasmid containing thymidine kinase (TK) and glycoproteins B (gB) genes of ILTV, fusion (F) and haemagglutinin-neuraminidase (HN) genes of NDV, membrane (M) and nucleocapsid (N) gene of IBV were designed and probe genes were amplified. Probes were purified using ethanol precipitation method and spotted on the amido-coating-glass slides to form a DNA microarray. Target genes were synthesized using fluorescent cy3-labeled primer by asymmetric PCR method and hybridized to microarray. Result of asymmetric PCR showed that single stranded of ILTV-TK, NDV-HN and IBV-N increased the most of all when concentration ratio of the restrictive and non-restrictive primer at 1∶10, and single product of ILTV-gB, NDV-F and IBV-M increased the most of all when the concentration ratio at 1∶20. The hybridization results showed the microarrays specific to the 3 poultry viruses could specifically hybridize with the corresponding sample, while any fluorescent signals could not be seen in negative controls. The sensitivity test showed that the concentration of DNA with 1.8×104 copies was still positive. Compared with the previous chip, hybridization efficiency and the hybridization signals increased significantly. Detection of 12 clinical samples, the results showed that the detection rate of DNA microarray and PCR method was consistent. This study showed that the developed microarray can simultaneously diagnosis 3 poultry disease of NDV-IBV-ILTV with fast, accurately and high-throughput, which can be applied to the detection a variety disease virus of chicken at intensive farming.
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Received: 27 April 2015
Published: 23 November 2015
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