| PCR结合斑点杂交技术检测禽多瘤病毒方法的建立及应用 |
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| 引用本文： | 张富友,于晓慧,蒋文明,孙淑红,赵鹏,刘华雷,李阳.PCR结合斑点杂交技术检测禽多瘤病毒方法的建立及应用[J].中国动物检疫,2021,38(3):92-98. |
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| 作者姓名： | 张富友 于晓慧 蒋文明 孙淑红 赵鹏 刘华雷 李阳 |
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| 作者单位： | 山东农业大学;中国动物卫生与流行病学中心 |
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| 摘 要： | 鹦鹉幼雏病是由禽类多瘤病毒(APV)引起的多种鹦鹉雏鸟死亡的急性病毒性传染病,严重危害鹦鹉养殖业的健康发展。为提高分子生物学方法检测APV的敏感性和特异性,对APV基因片段进行克隆和序列分析,设计合成1对特异性引物,以VP1基因为模板,经PCR扩增获得731 bp的核苷酸DNA,并用DIG标记,制备用于检测APV的特异性核酸探针;对制备的探针进行灵敏度检测,同时与普通PCR进行敏感性比较;使用制备的探针,对经分离鉴定和制备保存的其他7种禽病毒核酸进行特异性检测;用该核酸探针,对疑似感染APV的鹦鹉病料进行斑点杂交检测,并对鉴定为阳性的APV进行全基因组扩增和序列分析。结果显示:该探针可检测到2 pg量的APV特异性核酸片段;仅APV-VP1阳性核酸显色,呈现阳性反应,而阴性核酸和其他7种禽病毒核酸均不显色,呈阴性反应。结果表明:建立的核酸斑点杂交检测方法具有较高的灵敏度和特异性,可用于临床初步诊断。本方法的建立为我国开展APV分子流行病学调查及其感染的临床诊断提供了技术支撑。
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| 关 键 词： | 鹦鹉幼雏病 禽多瘤病毒 地高辛标记核酸探针 核酸斑点杂交 全基因组 遗传进化 |
Establishment and Application of PCR Combined with Dot Blot Hybridization for Detecting Avian Polyomavirus |
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| Zhang Fuyou,Yu Xiaohui,Jiang Wenming,Sun Shuhong,Zhao Peng,Liu Hualei,Li Yang.Establishment and Application of PCR Combined with Dot Blot Hybridization for Detecting Avian Polyomavirus[J].China Journal Of Animal Quarantine,2021,38(3):92-98. |
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| Authors: | Zhang Fuyou Yu Xiaohui Jiang Wenming Sun Shuhong Zhao Peng Liu Hualei Li Yang |
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| Institution: | (College of Veterinary Medicine,Shandong Agricultural University,Tai'an,Shandong 271018,China;China Animal Health and Epidemiology Center,Qingdao,Shandong 266114,China) |
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| Abstract: | Budgerigar fledgling disease(BFD)is an acute viral infectious disease caused by avian polyomavirus(APV),which could lead to death of various parrot nestlings and seriously endanger the healthy development of parrot industry.In order to improve the sensitivity and specificity of molecular biological methods to detect APV,the APV gene fragment was cloned and sequenced.After designing and synthesizing a pair of specific primers as well as carrying out PCR amplification,gene fragment with the nucleotide length of 731 bp was obtained by taking VP1 gene as a template,and then the fragment was marked with DIG to prepare a specific probe for detecting APV;the sensitivity of the probe was detected and compared with general PCR;other 7 kinds of isolated and prepared virus nucleic acids were used to evaluate the specificity of the probe;the lesions of parrots suspected to be infected with APV were detected by dot blot hybridization using the probe,and the whole genome sequences of the positive APV were amplified and analyzed.The results showed that the probe could detect 2 pg of APV specific nucleic acid fragment;and only the positive nucleic acids of APV-VP1 showed positive reaction,while the negative one and other 7 kinds of virus nucleic acids all showed negative reaction.In conclusion,the established method could be used for clinical preliminary diagnosis due to its high sensitivity and specificity.Therefore,molecular epidemiological investigation and clinical diagnosis of APV infection in China were provided with technical supports by the established method. |
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| Keywords: | BFD APV digoxigenin labled probe dot blot hybridization whole genome genetic evolution |
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