微流控芯片技术对禽流感病毒的快速检测
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摘要
为了快速、准确的检测H5、H7与H9亚型禽流感病毒,本研究利用反向斑点杂交方法和微流控芯片方法对禽流感病毒进行检测,初步建立了H5、H7与H9亚型禽流感病毒微流控芯片的鉴别诊断方法,并对微流控芯片和反向斑点杂交的检测灵敏度进行了比较研究。
本研究可分为三个部分:
第一部分:H5、H7与H9亚型禽流感病毒HA基因的克隆与序列分析
在H5与H9亚型禽流感病毒血凝素基因的保守区段内,选择设计2对引物,RT-PCR扩增后将目的片段连接到pGEM-T载体上,转化大肠杆菌感受态细胞,蓝白斑筛选,得到重组质粒pGEM-T-H5与pGEM-T-H9。重组质粒经限制性内切酶酶切、PCR及测序鉴定,结果证实试验用毒株分别为H5与H9亚型禽流感病毒。
H7亚型禽流感病毒HA片段参照Genbank上已发表的序列A/chicken/Italy/1285/2000(H7N1) (Genbank登录号: CY015014)人工合成,片段长208 bp (973~1 180 bp),片段连接到pMD18-T载体上,重组质粒命名为pMD18-T-H7。
第二部分:反向斑点杂交方法的建立
在H5、H7与H9亚型禽流感病毒血凝素基因的保守区段内,选择合成寡核苷酸探针与生物素标记引物。将探针通过紫外交联的方法固定在带正电荷的尼龙膜上,生物素标记引物用以扩增特异性片段。建立了能够同时扩增H5、H7与H9亚型禽流感病毒HA片段的多重RT-PCR方法,扩增产物经热变性后与探针42℃进行杂交,杂交后进行显色。出现明显的蓝紫色斑点判定为阳性,无斑点判定为阴性。对反向斑点杂交进行优化,整个杂交过程在RT-PCR后2.5 h内便可完成。该方法对H5与H9亚型禽流感病毒RNA的最低检测量分别为10 pg与1 pg;H7亚型可检测到1 pg质粒DNA。与琼脂糖凝胶电泳相比较,该方法检测灵敏度提高了10倍左右。3种亚型之间没有出现交叉反应,杂交特异性好。
使用Rheonix公司的Hyb室温杂交液,整个杂交在室温下即可完成,检测灵敏度与42℃条件下相当,且背景更浅。本试验建立的反向斑点杂交方法灵敏度高、特异性好,可以实现H5、H7与H9亚型禽流感病毒的鉴别诊断。
第三部分:微流控芯片快速检测H5、H7与H9亚型禽流感病毒方法的建立
在反向斑点杂交方法的基础上,建立了H5、H7与H9亚型禽流感病毒微流控芯片快速检测方法。微流控芯片将核酸提取、PCR反应、杂交集成在一起,实现了禽流感病毒的快速、准确、自动化检测。采用常规方法进行验证,证实该微流控芯片方法结果可靠。整个检测5 h内即可完成,H9亚型禽流感病毒RNA的最低检测量为1 pg,H5与H7亚型的最低检测量分别为10 pg与1 pg质粒DNA,检测灵敏度与反向斑点杂交方法相当。该方法检测效率高、试剂用量少、操作简便,不需要特殊仪器设备,易于在基层推广应用,可为我国禽流感的防控提供技术支持与保障。
The purpose of this study was to detect avian influenza virus (AIV) subtype H5, H7 and H9. The reverse dot blot hybridization assay and microfluidic chip method for identification of AIV subtype H5, H7 and H9 was established. The sensitivity of microfluidic chip method was compared with that of reverse dot blot hybridization assay.
The study consists of three parts:
Part I: Cloning and sequence analysis of HA gene of avian influenza virus subtype H5, H7 and H9
Two pairs of primers were designed according to the hemagglutinin’s conserved sequenses of AIV subtype H5 and H9. The interested fragments were amplified and inserted into a vector pGEM-T. Ligation products were transformed into E.coli competent cells. Doubtful strains were distinguished by blue-white spot screening, and then identified by restrictive endonucleonase, PCR and sequencing. Sequencing results showed that the cloned HA genes were successfully identified for AIV subtype H5 and H9. The recombinant plasmids were named as pGEM-T-H5 and pGEM-T-H9, respectively.
Based on the published hemagglutinin genes of AIV subtype H7 A/chicken/Italy/1285/2000(H7N1) (Genbank NO.: CY015014) in Genbank, 208 bp (973~1 180 bp) fragments were artifical synthesized,and then inserted into a pMD18-T vector. The recombinant plasmid was named as pMD18-T-H7。
PartⅡ: Development of reverse dot blot hybridization assay
Oligonucleotide probes and biotinylated primers were designed according to the conserved sequenses of hemagglutinin (HA) genes. The oligonucleotide probes were immobilized onto the positively charged nylon membrane by Ultraviolet (UV) radiation. A multiple RT-PCR was established with biotinylated primers for the amplification of HA fragment. Taget DNA fragments were amplified and then hybridized with oligonucleotide probes on the membrane at 42℃. Color reaction was carried out after hybridization. The presence of clearly visible purple-blue spots on the membrane was considered to be a positive hybridization reaction. The conditions of this assay was optimized, the whole procedure could be completed within 2.5h post-RT-PCR processing. 10 pg RNA of H5 subtype could be detected by reverse dot blot hybridization assay, and the detection limit of H9 subtype was 1pg RNA. 1 pg plasmid DNA of H7 subtype could be detected. The detection sensitivity of RDB was 10-fold higher than agarose gel electrophoresis. No cross-hybridization was detectecd between subtype H5, H7 and H9.
After using Rheonix’s RT Hyb hybridization solution, the hybridization could be done at room temperature, the detection sensitivity was equivalent with that of 42℃, and the background was lower. Reverse dot blot hybridization assay for identification of AIV subtype H5, H7 and H9 was specific and sensitive, which might become a rapid, effective method for identification of AIV.
PartⅢ:Establishment of the microfluidic chip method for detection of avian influenza virus subtype H5, H7 and H9
Based on reverse dot blot hybridization assay, the microfluidic chip method for identification of AIV subtype H5, H7 and H9 was established. Nucleic acid extration, PCR reaction and hybridization were integrated on one chip; AIV could be detected rapidly, accurately and automaticly. Verified by conventional methods, the results of microfluidic chip method were reliable. The whole procedure could be completed within 5 h; 1 pg RNA of H9 subtype could be detected by microfluidic chip method, the detection limit of H7 subtype was 1 pg plasmid DNA, and 10 pg plasmid DNA of H5 subtype could be detected. The detection sensitivity was equivalent with that of reverse dot blot hybridization.
The microfluidic chip method used fewer reagents, worked without special equipments under a higher efficiency. This assay will provide simple and effective technical support for rapid discrimination of AIV subtype H5, H7 and H9.
本研究可分为三个部分:
第一部分:H5、H7与H9亚型禽流感病毒HA基因的克隆与序列分析
在H5与H9亚型禽流感病毒血凝素基因的保守区段内,选择设计2对引物,RT-PCR扩增后将目的片段连接到pGEM-T载体上,转化大肠杆菌感受态细胞,蓝白斑筛选,得到重组质粒pGEM-T-H5与pGEM-T-H9。重组质粒经限制性内切酶酶切、PCR及测序鉴定,结果证实试验用毒株分别为H5与H9亚型禽流感病毒。
H7亚型禽流感病毒HA片段参照Genbank上已发表的序列A/chicken/Italy/1285/2000(H7N1) (Genbank登录号: CY015014)人工合成,片段长208 bp (973~1 180 bp),片段连接到pMD18-T载体上,重组质粒命名为pMD18-T-H7。
第二部分:反向斑点杂交方法的建立
在H5、H7与H9亚型禽流感病毒血凝素基因的保守区段内,选择合成寡核苷酸探针与生物素标记引物。将探针通过紫外交联的方法固定在带正电荷的尼龙膜上,生物素标记引物用以扩增特异性片段。建立了能够同时扩增H5、H7与H9亚型禽流感病毒HA片段的多重RT-PCR方法,扩增产物经热变性后与探针42℃进行杂交,杂交后进行显色。出现明显的蓝紫色斑点判定为阳性,无斑点判定为阴性。对反向斑点杂交进行优化,整个杂交过程在RT-PCR后2.5 h内便可完成。该方法对H5与H9亚型禽流感病毒RNA的最低检测量分别为10 pg与1 pg;H7亚型可检测到1 pg质粒DNA。与琼脂糖凝胶电泳相比较,该方法检测灵敏度提高了10倍左右。3种亚型之间没有出现交叉反应,杂交特异性好。
使用Rheonix公司的Hyb室温杂交液,整个杂交在室温下即可完成,检测灵敏度与42℃条件下相当,且背景更浅。本试验建立的反向斑点杂交方法灵敏度高、特异性好,可以实现H5、H7与H9亚型禽流感病毒的鉴别诊断。
第三部分:微流控芯片快速检测H5、H7与H9亚型禽流感病毒方法的建立
在反向斑点杂交方法的基础上,建立了H5、H7与H9亚型禽流感病毒微流控芯片快速检测方法。微流控芯片将核酸提取、PCR反应、杂交集成在一起,实现了禽流感病毒的快速、准确、自动化检测。采用常规方法进行验证,证实该微流控芯片方法结果可靠。整个检测5 h内即可完成,H9亚型禽流感病毒RNA的最低检测量为1 pg,H5与H7亚型的最低检测量分别为10 pg与1 pg质粒DNA,检测灵敏度与反向斑点杂交方法相当。该方法检测效率高、试剂用量少、操作简便,不需要特殊仪器设备,易于在基层推广应用,可为我国禽流感的防控提供技术支持与保障。
The purpose of this study was to detect avian influenza virus (AIV) subtype H5, H7 and H9. The reverse dot blot hybridization assay and microfluidic chip method for identification of AIV subtype H5, H7 and H9 was established. The sensitivity of microfluidic chip method was compared with that of reverse dot blot hybridization assay.
The study consists of three parts:
Part I: Cloning and sequence analysis of HA gene of avian influenza virus subtype H5, H7 and H9
Two pairs of primers were designed according to the hemagglutinin’s conserved sequenses of AIV subtype H5 and H9. The interested fragments were amplified and inserted into a vector pGEM-T. Ligation products were transformed into E.coli competent cells. Doubtful strains were distinguished by blue-white spot screening, and then identified by restrictive endonucleonase, PCR and sequencing. Sequencing results showed that the cloned HA genes were successfully identified for AIV subtype H5 and H9. The recombinant plasmids were named as pGEM-T-H5 and pGEM-T-H9, respectively.
Based on the published hemagglutinin genes of AIV subtype H7 A/chicken/Italy/1285/2000(H7N1) (Genbank NO.: CY015014) in Genbank, 208 bp (973~1 180 bp) fragments were artifical synthesized,and then inserted into a pMD18-T vector. The recombinant plasmid was named as pMD18-T-H7。
PartⅡ: Development of reverse dot blot hybridization assay
Oligonucleotide probes and biotinylated primers were designed according to the conserved sequenses of hemagglutinin (HA) genes. The oligonucleotide probes were immobilized onto the positively charged nylon membrane by Ultraviolet (UV) radiation. A multiple RT-PCR was established with biotinylated primers for the amplification of HA fragment. Taget DNA fragments were amplified and then hybridized with oligonucleotide probes on the membrane at 42℃. Color reaction was carried out after hybridization. The presence of clearly visible purple-blue spots on the membrane was considered to be a positive hybridization reaction. The conditions of this assay was optimized, the whole procedure could be completed within 2.5h post-RT-PCR processing. 10 pg RNA of H5 subtype could be detected by reverse dot blot hybridization assay, and the detection limit of H9 subtype was 1pg RNA. 1 pg plasmid DNA of H7 subtype could be detected. The detection sensitivity of RDB was 10-fold higher than agarose gel electrophoresis. No cross-hybridization was detectecd between subtype H5, H7 and H9.
After using Rheonix’s RT Hyb hybridization solution, the hybridization could be done at room temperature, the detection sensitivity was equivalent with that of 42℃, and the background was lower. Reverse dot blot hybridization assay for identification of AIV subtype H5, H7 and H9 was specific and sensitive, which might become a rapid, effective method for identification of AIV.
PartⅢ:Establishment of the microfluidic chip method for detection of avian influenza virus subtype H5, H7 and H9
Based on reverse dot blot hybridization assay, the microfluidic chip method for identification of AIV subtype H5, H7 and H9 was established. Nucleic acid extration, PCR reaction and hybridization were integrated on one chip; AIV could be detected rapidly, accurately and automaticly. Verified by conventional methods, the results of microfluidic chip method were reliable. The whole procedure could be completed within 5 h; 1 pg RNA of H9 subtype could be detected by microfluidic chip method, the detection limit of H7 subtype was 1 pg plasmid DNA, and 10 pg plasmid DNA of H5 subtype could be detected. The detection sensitivity was equivalent with that of reverse dot blot hybridization.
The microfluidic chip method used fewer reagents, worked without special equipments under a higher efficiency. This assay will provide simple and effective technical support for rapid discrimination of AIV subtype H5, H7 and H9.
引文
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