同时检测禽类六种病毒的金标银染可视化基因芯片的构建及初步应用

doi:10.11843/j.issn.0366-6964.2018.12.019

Abstract

Abstract:

The aim of the present study was to develop a visual chip for simultaneously detecting avian leukosis virus (ALV), chicken Marek's disease virus (MDV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), avian influenza virus (AIV), and infectious laryngotracheitis virus (ILTV) using gold label silver stain method. The gene sequences of ALV-env、MDV-meq and IBDV-vp₂ were cloned using T-A method. These sequences, also the sequences of AIV-np, NDV-f, ILTV-tk stored by our laboratory, were used for probes design, following which the simultaneously detecting ALV-MDV-IBDV-AIV-NDV-ILTV chip was developed. Following the PCR amplification using a pair of biotin-labeled primers, the chip was used for naked-eyes observation after hybridization and silver stain. The chip was then validated by specificity and sensitivity identification, stability evaluation, and clinical verification. The sequences of ALV-env, MDV-meq, IBDV-vp₂ were cloned successfully. The hybridization, at 40℃ for 2 h, of 50 μmol·L^-1 oligonucleotide probes with the biotin-labeled PCR products resulted in visible silver stain signal. No cross reaction was observed among ALV, MDV, IBDV, NDV, AIV and ILTV by specificity test. The specificity of the chip was confirmed by its negative hybridization with the PCR products from infectious bronchitis virus. The minimum-does of the plasmid as PCR template was 1 pg·μL^-1 by sensitivity test. The chip can be stored at room temperature for 60 d, or at 4℃ for 75 d according to stability test. The results of chip test consistent with that of PCR/RT-PCR method. The present study successfully developed a visual chip for simultaneously detecting ALV,MDV,IBDV,NDV,AIV and ILTV with an optimization reaction condition, which will facilitate the clinical application of the chip in the future.

CLC Number:

S858.315.3

XIANG Hua, YANG Guo-lin, CAO San-jie, HUANG Xiao-bo, WU Rui, ZHAO Qin, WEN Xin-tian, WEN Yi-ping. Development and Application of Gold Label Silver Stain Visual Chip for Simultaneously Detecting Six Kinds of Poultry Virus Diseases[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(12): 2698-2706.

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Development and Application of Gold Label Silver Stain Visual Chip for Simultaneously Detecting Six Kinds of Poultry Virus Diseases

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