H9N2亚型禽流感病毒HA基因体外转录及RNA标准品的初步构建
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摘要
为了提高H9N2亚型禽流感病毒核酸检测结果的准确性,同时减少试验操作中的污染,对H9N2亚型禽流感病毒HA全长基因序列进行克隆及体外转录,构建用于检测H9N2亚型禽流感HA基因阳性核酸标准品。采用实时荧光定量RT-PCR方法对转录的RNA片段进行验证。结果表明:HA体外转录产物可以准确提供RNA片段的拷贝数并进行定性和定量分析。H9N2亚型禽流感病毒HA基因体外转录RNA可作为H9N2禽流感病毒核酸检测标准品的候选产物。
In order to improve the accuracy of nucleic acid detection results of H9 N2 subtype avian influenza virus and reduce the pollution in the experiment,the HA full-length gene sequence of H9 N2 subtype avian influenza virus was cloned and in vitro transcribed to construct a HA-positive nucleic acid standard for H9 N2 subtype avian influenza. The transcription RNA fragment was validated by real-time fluorescence quantitative RT-PCR. The results showed that the copy number of the RNA fragment could be accurately provided by the extracellular transcription product of HA,and qualitative and quantitative analysis was carried out. Therefore,in vitro transcription RNA of H9 N2 subtype avian influenza virus HA gene can be used as a candidate for the detection of H9 N2 avian influenza virus nucleic acid.
In order to improve the accuracy of nucleic acid detection results of H9 N2 subtype avian influenza virus and reduce the pollution in the experiment,the HA full-length gene sequence of H9 N2 subtype avian influenza virus was cloned and in vitro transcribed to construct a HA-positive nucleic acid standard for H9 N2 subtype avian influenza. The transcription RNA fragment was validated by real-time fluorescence quantitative RT-PCR. The results showed that the copy number of the RNA fragment could be accurately provided by the extracellular transcription product of HA,and qualitative and quantitative analysis was carried out. Therefore,in vitro transcription RNA of H9 N2 subtype avian influenza virus HA gene can be used as a candidate for the detection of H9 N2 avian influenza virus nucleic acid.
引文
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[2]王伟利,周宇,赵丽,等.禽流感病毒H9N2亚型HA基因的序列分析[J].吉林农业大学学报,2006,28(1):84-87,92.
[3]孙泉云,陈琦,夏炉明.H9N2亚型禽流感流行现状[J].动物医学进展,2011,32(10):107-111.
[4]刘燕,钱爱东.禽流感病毒H5和H9亚型RT-PCR检测方法的建立[J].吉林农业大学学报,2004,26(1):93-96.
[5]杨素,廖秀云,廖明,等.禽流感病毒通用型及H5亚型套式荧光RT-PCR检测方法研究[J].中国畜牧兽医,2012,39(3):165-171.
[6]孙涛,雷质文,邓明俊,等.A型禽流感病毒通用型核酸检测标准样品的研制及不确定度分析[J].中国兽医学报,2014,34(9):1471-1475,1506.
[7]石火英,陈素娟,高崧,等.H9N2亚型禽流感病毒传播途径特性的比较及其表面基因序列分析[J].中国兽医学报,2007(1):26-30.
[8] Ha Y,Stevens D J,Skehel J J,et al. X-ray structures of H5 avian and H9 swine in fluenza virus hemagglutinins bound to avian and human receptor analogs[J]. Proceedings of the National Academy of Sciences,2001,98:11181-11186.
[9]孙文琪.2014-2016年华东地区H9N2亚型禽流感病毒遗传进化分析及气溶胶传播特性研究[D].扬州:扬州大学,2017.
[10]杨帆.禽流感检测技术研究进展[J].重庆工学院学报(自然科学版),2007(1):128-131,180.
[11]王坤,张玉静,孙兆增,等.H9N2亚型禽流感病毒NS1A融合基因的克隆表达及抗体的制备[J].吉林农业大学学报,2007,29(3):318-321.
[12]袁朋,许传田,张秀美,等.鸡干扰素α和白介素18在杆状病毒中共表达及其对H9N2禽流感抑制作用[J].吉林农业大学学报,2014,36(1):106-111.
[13]刘太峰,赵健乔,娄吉良,等.禽流感疫苗的研究进展[J].经济动物学报,2014,18(3):168-171,177.
[14]赵健乔,王军政,胡桂学,等.禽流感病毒的分子致病机理及其跨种传播的分子机制研究进展[J].经济动物学报,2014,18(3):178-183.
[15]丛彦龙,钟颖,孙艺学,等.H9N2亚型禽流感病毒流行病学及其疫苗的研究进展[J].中国兽医学报,2017,37(2):386-392.
[16]徐文章,孙艺学,邓效禹,等.禽H9N2亚型重组流感病毒疫苗候选株的构建及其免疫效果评价[J].中国兽医学报,2018,38(2):241-244.
[17]王潇,曹琛福,黄超华,等.禽流感病毒H5亚型RT-exoRPA检测方法的建立[J].中国兽医学报,2018,38(12):2339-2342,2347.
[18]刘海军. H9亚型禽流感病毒(重庆株)的分离鉴定及疫苗研究[D].重庆:重庆理工大学,2011.