A型流感病毒16种HA血清型DNA微阵列检测方法研究
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摘要
流感是巾流感病毒引起的具有高度传染性的呼吸系统疾病，流感病毒属于正黏病毒科，可分为A型，B型，C型。其中A型和B型流感病毒引起的季节性流感，发病率与死亡率高，造成的经济损失巨大，不断引发全球性健康问题。
A型流感病毒报据病毒表面蛋白血凝素（HA)和神经氨酸酶（NA)的抗原性，可分为17个HA和10个NA亚型，这些亚型的病毒组合几乎都可以从水生鸟类、家禽和其它禽类中分离到。A型流感病毒包含8个基因片段，至少编码14种以上的病毒蛋白。造成大流行的流感病毒常常是由两种毐株的不同RNA节段在感染细胞吋发生基因重排而产生的。流感病毒在理论上可以重组出现256(28)种不同基因型。
为应对流感流行，己出现了多种快速、灵敏、准确的分子生物学检测技术。II前对流感核酸样品的检测，大多数是基于聚合酶链反应（PCR)、DNA测序及寡核苷酸微阵列等三种技术。测序可以提供详细的序列，大范围的基因背景信息，也可检测出未知微生物，但这种方法费时，需昂贵设备，仍需人工去除其无关的背景DNA序列；RT-PCR/PCR检测技术是一种非常快速和灵敏的检测方法，但在检测多种微生物或对多种亚型进行分型时，仍受通量限制；而DNA微阵列技术恰好弥补了PCR及DNA测序技术的不足，且有良好的敏感性和特异性，还可实现高通蛍。
本研究计对当前A型流感病毒亚型多，高通量分型难等问题，应用低密度DNA微阵列分析技术，建立了流感病毒16种HA血清型鉴定方法。在NCBI流感病毒资源库中挑选出各亚型A型流感病毒代表序列，通过生物信息学方法分析16种亚型血凝素之间的同源关系，根据亚型间、亚型内的同源关系，确定探针的最佳长度，获得了每个亚型同--基因位点出现最多频率的…致序列，设计了18对PCR引物及108条用于分型鉴定的特异性寡核苷酸探针，实验筛选优选出54条探针。
在此基础上，将18对PCR引物设计分为4组，建立分组多重PCR方法，扩增及标记靶基因。通过体外转录方法制备16种HA基因的病毒RNA,梯度稀释，进行敏感性试验。经数据分析，最终确定当相应有…条探针SNR532為2或信号强度F532Mean-B532^2000时判定为阳性，得出本研究的最低检测下限在100-1000个RNA拷贝，并具有很好的重复性。
为验证DNA微阵列方法对新亚型流感病毒的检测性能，应用反向遗传操作技术拯救了重配H1N1、H4N6、H8N4、H9N1亚型流感病毒，并对其进行检测，结果表明，建立的检测方法可确定HI、H4、H8、H9等亚型反向遗传重配流感病毒株。
应用本研究室自有毒株对DNA微阵列检测方法进行了敏感性、特异性试验，可特异性检测H1N1、H3N2、H9N2、H5N1亚型等18种毒株，最低检测下限为1X103EID5Q病毒滴度。
幵展了流感分型DNA微阵列应急检测研究，成功回顾性检测了2004年吉林省白城市H5N1亚型高致性禽流感病毒阳性临床样品，并对2009年甲型H1N1流感病毒、季节性H1N1流感病毒和2013年H7N9亚型禽流感病毒进行区分鉴定，结果表明，研制的流感分型芯片可快速、特异并高通量筛查出不同亚型流感病毒，II在流感病毒应急防控中发挥了积极作用。
上述建立的DNA微阵列检测方法，能够准确进行A型、B型流感病毒及H1-16种亚型的分型检测，实现了检测流感病毒分型及亚型的高通量、高特异性，简化了复杂病原的操作程序，显著缩短了吋间，为流感快速检测、监测和应急处置提供了一个快速、方便、准确的分子生物学诊断技术。
Influenza is a highly contagious respiratory disease of humans, caused by negative-strand, segmented RNA viruses belonging to the family Orthomyxoviridae. There are three different genera of influenza virus; type A, type B, and type C. Seasonal outbreaks, caused by influenza A and B viruses, constitute a global health issue, leading to morbidity, mortality, and economic losses.
IAV is classified into17HA and10NA subtypes, based on the antigenicity of two viral surface proteins-hemagglutinin (HA) and neuraminidase (NA). Almost all possible combinations of HA and NA have been isolated from aquatic birds, poultry, and other avian species. In humans and other mammals, limited subtypes of IAVs have been detected. IAV contains eight gene segments encoding the corresponding viral protein(s). Pandemic viruses are generated by the rearrangement (reassortment) of viral RNA segments in cells infected with two different strains of IAV. Thus, reassortment can theoretically result in256(28) different genotypes, and is a key source of pandemic viruses. Avian and human IAVs can generally infect swine, and generate pandemic IAVs by reassortment. Molecular diagnostic techniques for viral testing have undergone rapid development in recent years, because of its rapid, sensitive and accurate advantages, more and more appropriate detection techniques applied to the field of molecular detection of influenza viruses, a variety of molecular diagnostic techniques have been widely used.
Most currently available methods for microbial detection and discovery using nucleic acid samples are based on three technologies. In order of increasing cost, these are the polymerase chain reaction (PCR), oligonucleotide microarrays and DNA sequencing. These platforms have different strengths and weaknesses. While sequencing provides the most in-depth, unbiased information, and is able to reveal completely novel organisms, it can be costly and time-consuming for some applications. Although multiplex sequencing of bar-coded samples reduces the cost per sample, it also decreases the coverage and thus the sensitivity of the analysis; this may be an issue when the organism of interest has low abundance and the sample has not been treated beforehand to remove host and/or background DNA.At the other end of the cost spectrum, PCR assays are very fast and sensitive, but have limited capacity for multiplexing.Microarrays occupy a middle ground with respect to cost, processing time, sensitivity, specificity and ability to detect novel organisms. Arrays can be designed with a combination of high-specificity probes and probes designed against conserved regions, so that they can be used in both detection and discovery modes.
On this basis, the18pairs of PCR primers were designed to be divided into four groups, to establish a packet multiplex PCR amplified and labeled target genes. Prepared by in vitro transcription16kinds of HA gene of the virus RNA, dilution for susceptibility testing. After data analysis, and ultimately determine if there is a corresponding probe SNR532≥2or signal strength F532Mean-B532≥2000judged as positive results of this study the minimum detection limit of RNA copies at100-1000, and has good repeatability.
To verify the DNA microarray method for the detection of a new influenza virus subtype performance, application reverse genetics rescued reassortant H1N1, H4N6, H8N4, H9N1subtype of influenza A virus, and its testing, the results show that the establishment of the detection method can be successfully identified H1, H4, H8, H9subtypes such as reverse genetics reassortant influenza virus strains.
The application of the laboratory's own strain of DNA microarray detection methods for sensitivity, specificity test, can specifically detect H1N1, H3N2, H9N2, H5N1subtype strains, etc., the minimum detection limit of1×103EIDso virus titers. Carried out DNA microarray genotyping influenza emergency detection research, the successful detection of a2004retrospective Baicheng City, Jilin caused by H5N1subtype avian influenza virus-positive clinical samples. And applied to the2009H1N1influenza virus detection and seasonal H1N1influenza virus to distinguish identification. The established method can be specifically identified2013H7N9subtype of avian influenza virus.
The developed application microarray detection method can be very accurately on the A-type, B-type influenza virus subtypes and H1-16for genotyping. Furthermore, the test results demonstrated that our system displays higher sensitivity and specificity than other methods. Collectively, this study provides a rapid, convenient and accurate molecular biology diagnostic technique for routine monitoring as well as for emergency responses to influenza pandemics in the future.
A型流感病毒报据病毒表面蛋白血凝素（HA)和神经氨酸酶（NA)的抗原性，可分为17个HA和10个NA亚型，这些亚型的病毒组合几乎都可以从水生鸟类、家禽和其它禽类中分离到。A型流感病毒包含8个基因片段，至少编码14种以上的病毒蛋白。造成大流行的流感病毒常常是由两种毐株的不同RNA节段在感染细胞吋发生基因重排而产生的。流感病毒在理论上可以重组出现256(28)种不同基因型。
为应对流感流行，己出现了多种快速、灵敏、准确的分子生物学检测技术。II前对流感核酸样品的检测，大多数是基于聚合酶链反应（PCR)、DNA测序及寡核苷酸微阵列等三种技术。测序可以提供详细的序列，大范围的基因背景信息，也可检测出未知微生物，但这种方法费时，需昂贵设备，仍需人工去除其无关的背景DNA序列；RT-PCR/PCR检测技术是一种非常快速和灵敏的检测方法，但在检测多种微生物或对多种亚型进行分型时，仍受通量限制；而DNA微阵列技术恰好弥补了PCR及DNA测序技术的不足，且有良好的敏感性和特异性，还可实现高通蛍。
本研究计对当前A型流感病毒亚型多，高通量分型难等问题，应用低密度DNA微阵列分析技术，建立了流感病毒16种HA血清型鉴定方法。在NCBI流感病毒资源库中挑选出各亚型A型流感病毒代表序列，通过生物信息学方法分析16种亚型血凝素之间的同源关系，根据亚型间、亚型内的同源关系，确定探针的最佳长度，获得了每个亚型同--基因位点出现最多频率的…致序列，设计了18对PCR引物及108条用于分型鉴定的特异性寡核苷酸探针，实验筛选优选出54条探针。
在此基础上，将18对PCR引物设计分为4组，建立分组多重PCR方法，扩增及标记靶基因。通过体外转录方法制备16种HA基因的病毒RNA,梯度稀释，进行敏感性试验。经数据分析，最终确定当相应有…条探针SNR532為2或信号强度F532Mean-B532^2000时判定为阳性，得出本研究的最低检测下限在100-1000个RNA拷贝，并具有很好的重复性。
为验证DNA微阵列方法对新亚型流感病毒的检测性能，应用反向遗传操作技术拯救了重配H1N1、H4N6、H8N4、H9N1亚型流感病毒，并对其进行检测，结果表明，建立的检测方法可确定HI、H4、H8、H9等亚型反向遗传重配流感病毒株。
应用本研究室自有毒株对DNA微阵列检测方法进行了敏感性、特异性试验，可特异性检测H1N1、H3N2、H9N2、H5N1亚型等18种毒株，最低检测下限为1X103EID5Q病毒滴度。
幵展了流感分型DNA微阵列应急检测研究，成功回顾性检测了2004年吉林省白城市H5N1亚型高致性禽流感病毒阳性临床样品，并对2009年甲型H1N1流感病毒、季节性H1N1流感病毒和2013年H7N9亚型禽流感病毒进行区分鉴定，结果表明，研制的流感分型芯片可快速、特异并高通量筛查出不同亚型流感病毒，II在流感病毒应急防控中发挥了积极作用。
上述建立的DNA微阵列检测方法，能够准确进行A型、B型流感病毒及H1-16种亚型的分型检测，实现了检测流感病毒分型及亚型的高通量、高特异性，简化了复杂病原的操作程序，显著缩短了吋间，为流感快速检测、监测和应急处置提供了一个快速、方便、准确的分子生物学诊断技术。
Influenza is a highly contagious respiratory disease of humans, caused by negative-strand, segmented RNA viruses belonging to the family Orthomyxoviridae. There are three different genera of influenza virus; type A, type B, and type C. Seasonal outbreaks, caused by influenza A and B viruses, constitute a global health issue, leading to morbidity, mortality, and economic losses.
IAV is classified into17HA and10NA subtypes, based on the antigenicity of two viral surface proteins-hemagglutinin (HA) and neuraminidase (NA). Almost all possible combinations of HA and NA have been isolated from aquatic birds, poultry, and other avian species. In humans and other mammals, limited subtypes of IAVs have been detected. IAV contains eight gene segments encoding the corresponding viral protein(s). Pandemic viruses are generated by the rearrangement (reassortment) of viral RNA segments in cells infected with two different strains of IAV. Thus, reassortment can theoretically result in256(28) different genotypes, and is a key source of pandemic viruses. Avian and human IAVs can generally infect swine, and generate pandemic IAVs by reassortment. Molecular diagnostic techniques for viral testing have undergone rapid development in recent years, because of its rapid, sensitive and accurate advantages, more and more appropriate detection techniques applied to the field of molecular detection of influenza viruses, a variety of molecular diagnostic techniques have been widely used.
Most currently available methods for microbial detection and discovery using nucleic acid samples are based on three technologies. In order of increasing cost, these are the polymerase chain reaction (PCR), oligonucleotide microarrays and DNA sequencing. These platforms have different strengths and weaknesses. While sequencing provides the most in-depth, unbiased information, and is able to reveal completely novel organisms, it can be costly and time-consuming for some applications. Although multiplex sequencing of bar-coded samples reduces the cost per sample, it also decreases the coverage and thus the sensitivity of the analysis; this may be an issue when the organism of interest has low abundance and the sample has not been treated beforehand to remove host and/or background DNA.At the other end of the cost spectrum, PCR assays are very fast and sensitive, but have limited capacity for multiplexing.Microarrays occupy a middle ground with respect to cost, processing time, sensitivity, specificity and ability to detect novel organisms. Arrays can be designed with a combination of high-specificity probes and probes designed against conserved regions, so that they can be used in both detection and discovery modes.
On this basis, the18pairs of PCR primers were designed to be divided into four groups, to establish a packet multiplex PCR amplified and labeled target genes. Prepared by in vitro transcription16kinds of HA gene of the virus RNA, dilution for susceptibility testing. After data analysis, and ultimately determine if there is a corresponding probe SNR532≥2or signal strength F532Mean-B532≥2000judged as positive results of this study the minimum detection limit of RNA copies at100-1000, and has good repeatability.
To verify the DNA microarray method for the detection of a new influenza virus subtype performance, application reverse genetics rescued reassortant H1N1, H4N6, H8N4, H9N1subtype of influenza A virus, and its testing, the results show that the establishment of the detection method can be successfully identified H1, H4, H8, H9subtypes such as reverse genetics reassortant influenza virus strains.
The application of the laboratory's own strain of DNA microarray detection methods for sensitivity, specificity test, can specifically detect H1N1, H3N2, H9N2, H5N1subtype strains, etc., the minimum detection limit of1×103EIDso virus titers. Carried out DNA microarray genotyping influenza emergency detection research, the successful detection of a2004retrospective Baicheng City, Jilin caused by H5N1subtype avian influenza virus-positive clinical samples. And applied to the2009H1N1influenza virus detection and seasonal H1N1influenza virus to distinguish identification. The established method can be specifically identified2013H7N9subtype of avian influenza virus.
The developed application microarray detection method can be very accurately on the A-type, B-type influenza virus subtypes and H1-16for genotyping. Furthermore, the test results demonstrated that our system displays higher sensitivity and specificity than other methods. Collectively, this study provides a rapid, convenient and accurate molecular biology diagnostic technique for routine monitoring as well as for emergency responses to influenza pandemics in the future.
引文
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