| 猪流感病毒H1N1、H1N2和H3N2亚型多重RT-PCR诊断方法的建立 |
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| 引用本文： | 杨焕良,乔传玲,陈艳,辛晓光,李一经,陈化兰.猪流感病毒H1N1、H1N2和H3N2亚型多重RT-PCR诊断方法的建立[J].中国预防兽医学报,2007,29(9):714-718. |
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| 作者姓名： | 杨焕良 乔传玲 陈艳 辛晓光 李一经 陈化兰 |
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| 作者单位： | 1. 中国农业科学院哈尔滨兽医研究所,农业部动物流感重点开放实验室/兽医生物技术国家重点实验室,黑龙江,哈尔滨,150001
2. 东北农业大学,动物医学院,黑龙江,哈尔滨,150030 |
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| 摘 要： | 对我国分离到的猪流感病毒和GenBank数据库中已有的猪流感病毒H1N1、H1N2和H3N2亚型毒株的HA、NA基因核苷酸序列进行分析,分别选出各个病毒亚型HA和NA基因中高度保守且特异的核苷酸区域,设计扩增猪流感病毒H1和H3、N1和N2亚型的2套多重PCR特异性引物,建立了猪流感H1N1、H1N2和H3N2亚型病毒多重RT-PCR诊断方法。采用该方法对H1N1、H1N2、H3N2亚型猪流感病毒标准参考株进行RT-PCR检测,结果均呈阳性,对扩增得到的片段进行序列测定和BLAST比较,表明为目的基因片段。其它几种常见猪病病毒和其它亚型猪流感病毒的RT-PCR扩增结果都呈阴性。对107EID50/0.1mL病毒进行稀释,提取RNA进行敏感性试验,RT-PCR最少可检测到102EID50的病毒量核酸。对40份阳性临床样品的检测结果是H1N1、H1N2和H3N2亚型分别为16份、1份和20份,其它3份样品同时含有H1N1和H3N2亚型猪流感病毒,和鸡胚分离病毒结果100%一致。试验证明建立的猪流感病毒H1N1、H1N2和H3N2亚型多重RT-PCR诊断方法是一种特异敏感的诊断方法,可用于临床样品的早期快速诊断和分型。
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| 关 键 词： | 猪流感 病毒 多重RT-PCR |
| 文章编号： | 1008-0589（2007）09-0714-04 |
| 修稿时间： | 2007-01-23 |
Subtyping of H1N1, H1N2 and H3N2 swine influenza viruses by two multiplex RT-PCR |
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| YANG Huan-liang,QIAO Chuan-ling,CHEN Yan,XIN Xiao-guang,LI Yi-jing,CHEN Hua-lan.Subtyping of H1N1, H1N2 and H3N2 swine influenza viruses by two multiplex RT-PCR[J].Chinese Journal of Preventive Veterinary Medicine,2007,29(9):714-718. |
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| Authors: | YANG Huan-liang QIAO Chuan-ling CHEN Yan XIN Xiao-guang LI Yi-jing CHEN Hua-lan |
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| Institution: | 1. Key Laboratory of Animal Influenza, Ministry of Agriculture, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150001, China; 2. College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China |
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| Abstract: | Two multiplex RT-PCR assays specific for influenza virus H1 and H3,N1 and N2 subtypes were developed.Primers were designed from conserved regions of the HA and NA genes of swine influenza H1N1,H1N2 and H3N2 viruses according to the published sequences in GenBank and sequences from China isolates of swine influenza virus.The RT-PCR amplified successfully the HA and NA genes of H1N1,H1N2 and H3N2 subtype of swine influenza viruses.Sequence analysis of PCR products by BLAST search revealed the highest identity among viruses of the same HA and NA subtype and showed complete correlation with conventional typing methods.The assays were highly specific and did not amplify PCR products from 3 common swine pathogens or influenza viruses other than reference strains;and have a sensitivity of detecting RNA extracted from up to 102 EID50 of reference virus(with original infectivity titer of 107 EID50/0.1 mL).Test against 40 positive samples showed that swine influenza virus H1N1,H1N2 and H3N2 were identified in 16,1 and 20 samples,respectively,while the remaining 3 samples were detected positive for both H1N1 and H3N2 viruses.These results were 100 % in agreement with those of virusisolated in embryonated eggs.Therefore the multiplex RT-PCR assays could be an effective tool for rapid detection and simultaneous subtyping of clinical swine influenza virus. |
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| Keywords: | H1N1 H1N2 H3N2 |
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