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| 双重RT-PCR同时快速检测H5亚型禽流感病毒和新城疫病毒强毒株 | |
| 引用本文： | 张斌,汤承,张兆敏,马莉,刘小银,李明义,岳华.双重RT-PCR同时快速检测H5亚型禽流感病毒和新城疫病毒强毒株[J].中国动物检疫,2009,26(2):24-28. |
| 作者姓名： | 张斌 汤承 张兆敏 马莉 刘小银 李明义 岳华 |
| 作者单位： | 1. 西南民族大学生命科学与技术学院,四川成都,610041 2. 中国医学科学院成都输血研究所,四川成都,610081 3. 四川大学华西第二医院发育与干细胞生物学研究所,成都,610041 4. 中国动物卫生与流行病学中心,山东青岛,266032 |
| 摘 要： | 本研究针对AIV H5的血凝素(HA)基因和vNDV的融合蛋白(F)基因设计两对引物,建立了单管同时检测AIVH5和vNDV的双重RT-PCR(dRT-PCR),该方法能在单一反应管内同时检测AIVH5和vNDV,不与其它亚型AIV、弱毒NDV毒株以及其它病原体发生非特异性扩增;对含有AIV H5 HA基因和vNDV F基因重组质粒DNA的最低检测限分别为20.6和406fg,敏感性与单项RT-PCR相同;从样本处理到报告结果仅需5h。对24份临床疑似样本进行检测,AIVH5均为阴性,vNDV阳性18份,PCR产物测序证明为靶基因序列,其具有vNDV F基因的特征性序列,随机选9份vNDV阳性样本接种SPF鸡胚,分离出6株vNDV,病毒分离率为6/9;对100 ELD50的AIV H5N1和100 ELD50的vNDV人工同时感染5日龄SPF鸡的脑、肝脏、肺脏和泄殖腔棉拭子进行dRT-PCR检测,AIV H5的检测率为4/5,3/5,5/5,4/5,vNDV的检测率为3/5,5/5,4/5,3/5,而对H9N2和LaSota毒株实验同时感染样本及阴性对照样本的检测均为阴性。可见本研究建立的dRT-PCR为AIV H5和vNDV诊断、监测、检疫及分子流行病学调查提供了特异性强、操作简单、检测速度快、成本低廉的新方法。 |
| 关 键 词： | 禽流感H5亚型 强毒力新城疫 双重RT-PCR 快速检测 |
The duplex RT-PCR Assay for Simultaneous Detection of H5 Subtype Avian Influenza Viruses and Velogenic Newcastle Disease virus |
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| ZHANG Bin,TANG Cheng,ZHANG Zhaomin,MA Li,LIU Xiaoyin,LI Mingyi,YUE Hua.The duplex RT-PCR Assay for Simultaneous Detection of H5 Subtype Avian Influenza Viruses and Velogenic Newcastle Disease virus[J].China Journal Of Animal Quarantine,2009,26(2):24-28. | |
| Authors: | ZHANG Bin TANG Cheng ZHANG Zhaomin MA Li LIU Xiaoyin LI Mingyi YUE Hua |
| Institution: | 1 College of Life Science and Technology, Southwest University for Nationalities, Chengdu, 610041; 2 Institute of transfusion Chinese academy of medical sciences, Chengdu,610081; 3 West China Institute of Developmental & Stem Cell Biology West China Women’s & Children’s Hospital Sichuan University 4.China Animal and Epidemiology Center,Qingdao,266032 |
| Abstract: | A duplex polymerase chain reaction assay(dRT-PCR)was developed in this study which could simultaneously ampli- fy the hemagglutinin glycoprotein (HA) gene of the AIV H5 and distinctive sequence of vNDV F gene in a single tube. The positive results could be obtained from AIV H5 and vNDV strains while no positive amplification was observed with other HA subtypes of AIV, avirulent NDV and other unrelated avian pathogens. The endpoint of detection was defined as approxi- mately 20.6fg for DNA plasmid containing HA gene of AIV H5 and 406fg for DNA plasmid containing F gene of vNDV. Compared with the single RT-PCR, the dRT-PCR assay was able to detect AIV/NDV with similar sensitivity. The whole process of detection, from sample processing to obtaining the results, could be finished within 5h. Of 24 clinical samples detected by this assay, all were AIV-H5 negative and 18 were vNDV positive. Sequencing analysis confirmed that all PCR products contained characteristic sequence of vNDV at cleavage site. NDV isolation was performed by SPF embryo from 9 clinical sam- ples randomly selected and showed a 6/9 isolation rate. Samples of livers, brains, lungs and cloacal swabs collected from 5 five-day-old SPF chicken experimentally co-infected with 100ELD50 AIV H5 and 100ELD50 vNDV were detected by the dRT-PCR, the detection rate of AIV H5 was 4/5,3/5,5/5, 4/5, the detection rate of vNDV was 3/5,5/5,4/5,3/5; while no positive samples from chicken experimentally co-infected with AIV H9N2 and LaSota, and negative control has been found using the dRT-PCR, In conclusion, the method developed in this study provides a rapid, accurate, economical and effective way to detect AIV H5 and vNDV. |
| Keywords: | Subtype H5 avian influenza virus velogenic Newcastle disease virus duplex RT-PCR rapid detection |
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