猪流感病毒分型基因芯片的研制与应用
摘要
猪流感(Swine influenza,SI)是由正黏病毒科猪流感病毒(SIV)引起的一种急性、高度接触传染性的群发性猪呼吸道疾病,目前已发现的SIV至少有H1N1、H1N2、H1N7、H3N2、H3N6、H4N6、H9N2和H5N1等8种不同血清亚型。血凝抑制试验(HI)和神经氨酸酶抑制试验(NI)是流感病毒亚型鉴定的常用方法,另外琼脂扩散试验、酶联免疫吸附试验等其他血清学方法也用于猪流感病毒亚型的检测,但是这些方法都存在灵敏度低且经验依赖性强等特点。PCR是一种快速准确的检测方法,但是普通的PCR方法只能一次对一种亚型进行检测,不能满足实际检测的需要。
本研究根据流感病毒的HA和NA基因序列间的差异性,采用基因芯片技术,借助多重PCR和核酸标记技术,构建了猪流感病毒亚型分型基因芯片,用于猪流感病毒亚型的分型,具有检测A型流感病毒和同步鉴别猪流感病毒H1、H3、H9、N1和N2亚型的功能。本研究共分为以下3部分:
1猪流感病毒分型基因芯片的建立
本部分主要包括芯片的设计和制备、探针浓度和Biotin-11-dUTP使用浓度的选择,杂交时间和杂交温度的优化,芯片的灵敏度、特异性、重复性和稳定性检测。
根据Genbank发表的核酸序列,运用DNAstar软件,分析流感病毒的HA和NA基因序列间的差异性,分别设计合成流感病毒H1、H3、H9、N1和N2的特异分型引物,并根据A型流感病毒的型特异性基因(M基因),设计了A型流感病毒的通用引物。应用RT-PCR分别扩增各亚型的特异片断,并分别克隆到pMD18-T Simple Vector构建重组质粒,用于各亚型探针的制备。将各探针测序后,应用BLAST对扩增产物测序结果与相应病毒亚型核酸序列的进行同源比对,证明与相应亚型的大部分毒株同源性在90%以上。将各探针纯化后,统一稀释为75 ng/μL,按一定的阵列点加到硝酸纤维素膜上,80℃烤膜2 h,制备成低密度基因芯片,并对芯片制备和杂交条件的优化以及芯片的性能进行测定。结果筛选出制备芯片的Biotin-11-dUTP使用浓度为80μM,探针浓度为75 ng/μL,杂交温度为42℃,杂交时间1 h。制备的芯片具有良好的特异性和可重复性,灵敏度比PCR高1个稀释度,比病毒分离高2个稀释度。该方法的建立,为猪流感的检测及猪流感病毒各亚型在猪群中的流行病学调查提供了一种快速、灵敏和高通量的手段。
2不对称PCR在基因芯片中的应用
利用不对称PCR可以制备单链PCR产物,较少基因芯片杂交时自身杂交的损失,提高基因芯片的杂交率,但同时也会减少PCR的最终产物。本试验选择了1:25、1:50、1:100和1:200的稀释滴度,芯片对比检测结果显示,在引物浓度为1:100时杂交结果最好,与不用不对称PCR的芯片检测结果相比,灵敏度提高了1～2个稀释度。
3猪流感病毒分型基因芯片的初步应用
利用制备好的基因芯片检测了从青岛、烟台、潍坊、济宁、南昌、葫芦岛等市健康猪群或病死猪采集的鼻拭子或组织426份,检测结果和PCR的符合率在87%以上,鸡胚传代病毒分离检测结果同基因芯片与PCR的检测结果相差比较大。结果表明猪流感病毒分型基因芯片可以作为猪流感病毒监测的一种重要手段,其灵敏度高于PCR方法。
Swine Influenza is an acute, high contagiousness respiratory disease of swine with a type A Influenza virus which belongs to the Orthomyxoviridae family. At present, eight subtypes of Swine Influenza Virus(SIV) which have been isolated are H1N1, H1N2, H1N7, H3N2, H3N6, H4N6, H9N2 and H5N1, the frequently used methods for identifying subgroups Influenza virus are hemagglutination inhibition assay(HI) and neuramidinase inhibition assay (NI). Besides, the agar diffusion reaction, enzyme linked immunosorbent assay and other serological methods can identify subgroups Influenza virus. Whereas these methods are all lower sensitivtive and hadro-dependence by experience. PCR is a quick and precise method, while the vukgar PCR only test one subtype once. Therefore it can not satisfy the need of detection.
This research constructs low density DNAarray for diagnosing the subtypes of SIV by using the technology of DNAarray, multiplicitas PCR and nucleinic acid mark, which can diagnose the subtypes of SIV and the type A Influenza virus at the same time. Major studies are processed as follows:
Part 1: Establishment of Low Density DNAarray for Diagnosing the Subtypes of SIV This part includes probe concentration options, biotin-11-dUTP concentration options, hybridization temperature and hybridization time options, sensitivity, specificity, reproducibility and constant testing.
The hemagglutinin(HA) and neuraminidase(NA) gene of five subtypes influenza A virus were amplified by RT-PCR with primers based on the diversity of HA and NA gene. Meanwhile, conserved cDNA fragments of M were amplified by RT-PCR with consensus primer. Each fragment was cloned in pMD18-T Simple Vectors to construct recombinant plasmid respectively, and constructed recombinant plasmid for DNA probe preparation and recovered DNA probes were spotted on located sites on nitrocellulose filter to be DNAarray. After sequencing, the result was tested by using BLAST and compared with the corresponding subetypes. Aliment reports show that the sequence is above 90% with the bulk corresponding subetypes. After purificating, the probes are diluted to 75 ng/μ, and marked nitrocellulose filter, fixed to two hours, determined the preparation of genechip and condition of hybridization. The results show that the Biotin-11-dUTP is 80μM, probe is 75 ng/μl; hybridization temperature is 42℃, hybridization time is 1 hour. These DNAarrays have specificity and good reproducitivity. The sensitivity of genechip is one dilution higher than PCR, and two dilution higher than viral isolation. These DNAarray provids a quick, sensitive and high-flux method for large-scale epidemiology investigation and quarantine of Swine Influenza Virus.
Part 2: Application of dissymmetry PCR in DNAarray
Dissymmetry PCR can be applied to prepare single strand PCR products, and that can reduce the lose of self-hybridization in DNAarray hybridization. It raises the hybridization rate, and reduces the final prducts of PCR. This research chooses 1:25, 1:50, 1:100 and 1:200 titre. It shows that the optimization primer concentration is 1:100, the sensitivity is raised 10～100 times comparing with the conventional PCR.
Part 3: The primary application of DNAarray in diagnosing the subtypes of SIV
The DNAarray is applied to detect the nose swab or tissue samples of health swine or illness swine from Qingdao、Yantai、Weifang、Jining、Nanchang、Huludao and so on. The result shows that the coincidence of DNAarray and PCR is above 87%, but it has significant diversity with the method in which the virus was isolated by embryo passage. It shows that this DNAarray can be applied to monitor the swine influenza, the sensitivity is higher than PCR.
本研究根据流感病毒的HA和NA基因序列间的差异性,采用基因芯片技术,借助多重PCR和核酸标记技术,构建了猪流感病毒亚型分型基因芯片,用于猪流感病毒亚型的分型,具有检测A型流感病毒和同步鉴别猪流感病毒H1、H3、H9、N1和N2亚型的功能。本研究共分为以下3部分:
1猪流感病毒分型基因芯片的建立
本部分主要包括芯片的设计和制备、探针浓度和Biotin-11-dUTP使用浓度的选择,杂交时间和杂交温度的优化,芯片的灵敏度、特异性、重复性和稳定性检测。
根据Genbank发表的核酸序列,运用DNAstar软件,分析流感病毒的HA和NA基因序列间的差异性,分别设计合成流感病毒H1、H3、H9、N1和N2的特异分型引物,并根据A型流感病毒的型特异性基因(M基因),设计了A型流感病毒的通用引物。应用RT-PCR分别扩增各亚型的特异片断,并分别克隆到pMD18-T Simple Vector构建重组质粒,用于各亚型探针的制备。将各探针测序后,应用BLAST对扩增产物测序结果与相应病毒亚型核酸序列的进行同源比对,证明与相应亚型的大部分毒株同源性在90%以上。将各探针纯化后,统一稀释为75 ng/μL,按一定的阵列点加到硝酸纤维素膜上,80℃烤膜2 h,制备成低密度基因芯片,并对芯片制备和杂交条件的优化以及芯片的性能进行测定。结果筛选出制备芯片的Biotin-11-dUTP使用浓度为80μM,探针浓度为75 ng/μL,杂交温度为42℃,杂交时间1 h。制备的芯片具有良好的特异性和可重复性,灵敏度比PCR高1个稀释度,比病毒分离高2个稀释度。该方法的建立,为猪流感的检测及猪流感病毒各亚型在猪群中的流行病学调查提供了一种快速、灵敏和高通量的手段。
2不对称PCR在基因芯片中的应用
利用不对称PCR可以制备单链PCR产物,较少基因芯片杂交时自身杂交的损失,提高基因芯片的杂交率,但同时也会减少PCR的最终产物。本试验选择了1:25、1:50、1:100和1:200的稀释滴度,芯片对比检测结果显示,在引物浓度为1:100时杂交结果最好,与不用不对称PCR的芯片检测结果相比,灵敏度提高了1～2个稀释度。
3猪流感病毒分型基因芯片的初步应用
利用制备好的基因芯片检测了从青岛、烟台、潍坊、济宁、南昌、葫芦岛等市健康猪群或病死猪采集的鼻拭子或组织426份,检测结果和PCR的符合率在87%以上,鸡胚传代病毒分离检测结果同基因芯片与PCR的检测结果相差比较大。结果表明猪流感病毒分型基因芯片可以作为猪流感病毒监测的一种重要手段,其灵敏度高于PCR方法。
Swine Influenza is an acute, high contagiousness respiratory disease of swine with a type A Influenza virus which belongs to the Orthomyxoviridae family. At present, eight subtypes of Swine Influenza Virus(SIV) which have been isolated are H1N1, H1N2, H1N7, H3N2, H3N6, H4N6, H9N2 and H5N1, the frequently used methods for identifying subgroups Influenza virus are hemagglutination inhibition assay(HI) and neuramidinase inhibition assay (NI). Besides, the agar diffusion reaction, enzyme linked immunosorbent assay and other serological methods can identify subgroups Influenza virus. Whereas these methods are all lower sensitivtive and hadro-dependence by experience. PCR is a quick and precise method, while the vukgar PCR only test one subtype once. Therefore it can not satisfy the need of detection.
This research constructs low density DNAarray for diagnosing the subtypes of SIV by using the technology of DNAarray, multiplicitas PCR and nucleinic acid mark, which can diagnose the subtypes of SIV and the type A Influenza virus at the same time. Major studies are processed as follows:
Part 1: Establishment of Low Density DNAarray for Diagnosing the Subtypes of SIV This part includes probe concentration options, biotin-11-dUTP concentration options, hybridization temperature and hybridization time options, sensitivity, specificity, reproducibility and constant testing.
The hemagglutinin(HA) and neuraminidase(NA) gene of five subtypes influenza A virus were amplified by RT-PCR with primers based on the diversity of HA and NA gene. Meanwhile, conserved cDNA fragments of M were amplified by RT-PCR with consensus primer. Each fragment was cloned in pMD18-T Simple Vectors to construct recombinant plasmid respectively, and constructed recombinant plasmid for DNA probe preparation and recovered DNA probes were spotted on located sites on nitrocellulose filter to be DNAarray. After sequencing, the result was tested by using BLAST and compared with the corresponding subetypes. Aliment reports show that the sequence is above 90% with the bulk corresponding subetypes. After purificating, the probes are diluted to 75 ng/μ, and marked nitrocellulose filter, fixed to two hours, determined the preparation of genechip and condition of hybridization. The results show that the Biotin-11-dUTP is 80μM, probe is 75 ng/μl; hybridization temperature is 42℃, hybridization time is 1 hour. These DNAarrays have specificity and good reproducitivity. The sensitivity of genechip is one dilution higher than PCR, and two dilution higher than viral isolation. These DNAarray provids a quick, sensitive and high-flux method for large-scale epidemiology investigation and quarantine of Swine Influenza Virus.
Part 2: Application of dissymmetry PCR in DNAarray
Dissymmetry PCR can be applied to prepare single strand PCR products, and that can reduce the lose of self-hybridization in DNAarray hybridization. It raises the hybridization rate, and reduces the final prducts of PCR. This research chooses 1:25, 1:50, 1:100 and 1:200 titre. It shows that the optimization primer concentration is 1:100, the sensitivity is raised 10～100 times comparing with the conventional PCR.
Part 3: The primary application of DNAarray in diagnosing the subtypes of SIV
The DNAarray is applied to detect the nose swab or tissue samples of health swine or illness swine from Qingdao、Yantai、Weifang、Jining、Nanchang、Huludao and so on. The result shows that the coincidence of DNAarray and PCR is above 87%, but it has significant diversity with the method in which the virus was isolated by embryo passage. It shows that this DNAarray can be applied to monitor the swine influenza, the sensitivity is higher than PCR.
引文
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