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| The Optimization and Preliminary Application of Visual Microarray for Detecting Four Respiratory Disease of Chicken (Gallus gallus) |
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Abstract Visual microarray technology based on the traditional microarray technology, has a great significance for the detection of clinical disease detection and diagnosis. In this study, the specific primers were designed, according to the conservative gene nucleprotein (NP) of Avian influenza virus (AIV), cDNA was obtained from RT-PCR. The recombinant plasmid of AIV-NP was successfully constructed. Meanwhile, fusion (F) gene of Newcastle disease virus (NDV), nucleocapsid (N) gene of Infectious bronchitis virus (IBV) and thymidine kinase (TK) gene of Infectious laryngotracheitis virus (ILTV) were successfully recovered from the conserved bacteria. The oligonucleotide probes were designed on the basis of target gene sequences. The visual microarray was prepared and hybridized to the PCR products labeled biotin. The parameters of the visual microarray were optimized. The results showed that 25 μmol/L of oligonucleotide probes, 1 h of hybridization time, 50 ℃ of hybridization temperature, 2 000 times diluted of 1.0 mg/mL Streptavidin HRP Conjugate and 5 min of diaminobenzidine (DAB) staining time, were the optimal condition for microarray detection. The 96 clinical samples were simultaneously detected by the visual microarray and PCR/RT-PCR, the results showed that the detection rate of two methods was consensus. The visual microarray of AIV-NDV-IBV-ILTV developed in this study is a fast, accurately and high-throughput detection methods, which could provide a new technology for chicken disease clinical diagnosis.
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Received: 14 June 2017
Published: 14 February 2018
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