| 虎源流感病毒RT-PCR检测方法的建立及其应用研究 |
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| 作者姓名： | 贺文琦 高玉伟 夏咸柱 杨松涛 王立刚 刘 丹 |
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| 作者单位： | 军事医学科学院军事兽医研究所，吉林大学畜牧兽医学院 吉林长春130062，军事医学科学院军事兽医研究所，军事医学科学院军事兽医研究所，军事医学科学院军事兽医研究所，黑龙江省东北虎林园，黑龙江省东北虎林园，吉林长春130062，吉林长春130062，吉林长春130062，吉林长春130062，黑龙江哈尔滨150027，黑龙江哈尔滨150027 |
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| 基金项目： | 国家自然科学基金资助项目(30471Z95)，全军医学科研基金资助项目(04Z016)，国家科技部科技攻关计划课题(2004BA519A11) |
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| 摘 要： | 为临床上能快速诊断虎流感,通过对已分离获得的虎源H5N1流感病毒和GenBank中报道的A型H5N1亚型流感病毒的NP、HA、NA序列,设计合成各个基因的特异性PCR扩增引物和A型流感病毒通用反转录引物。通过优化反应体系及条件,建立一步法可同时扩增出3个基因的联合RT-PCR检测方法。将该方法用于临床病料检测,并与电镜负染、HA/HI及病毒分离等方法进行比较。结果,NP、HA、NA基因的扩增片段大小分别为464bp、581bp、173bp,其检测的敏感性约100个TCID50。该方法的检出率和病毒分离结果相符,高于电镜负染和HA/HI试验。结果显示,该方法可用于虎源流感病毒的临床或实验室检测。
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| 关 键 词： | 虎源流感病毒 H5N1 RT-PCR 检测方法 |
Research of a RT-PCR Assay for Multiple Genes Detection on Tiger Influenza Virus |
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| Authors: | He Wenqi Gao Yuwei Xia Xianzhu Yang Songtao Wang Ligang Liu Dan |
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| Institution: | 1.The Military Veterinary Institute, Academy of Military Medical Scineces, Changchun 130062; 2.The Veterinary Institute of Jilin University, Changchun 130062; 3.Heilongjiang Siberia Tiger Park, Harbin 150027 |
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| Abstract: | In order to diagnose quickly tiger influenza in clinic, using the tiger influenza virus (TIV) sequence and the influenza A virus sequence information available in the Genbank data base, the primers were selected from conserved region of a target gene specific for TIV type (NP) and subtype (HA,NA) for use in RT-PCR. Specific primers were desiged for NP, HA, NA gene with Primer5.0 primer design software, DNA fragments of sizes 464, 581 and 173 bp, respectively, were amplified. The primers to be in common used for influenza A virus were desiged for use in the synthesis of cDNAs. Optimize the reaction condition and establish a multiplex RT-PCR in a single step for simultaneous detection of the TIV 3 genes fragments. The assay were applied in clinical diagnosis. This decision was made following the results of a comparison with electron microscope, hemagglutination /hemagglutination inhibition and isolation of the virus in tissue culture or allantoic fluid. The results were that the sensitivity of detection was about 100 TCID50, an excellent concordance between diagnosis by the RT-PCR and culture was demonstrated, and the RT-PCR in detection of TIV is more sensitive and rapid than above mentioned methods. So the method described here is very practicable and valuable in clinical diagnosis or in laboratory application. |
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| Keywords: | Tiger influenza virus H5N1 RT-PCR Detecting method |
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