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| 基于荧光熔解曲线模式RT-PCR快速检测H5N1亚型禽流感病毒 | |
| 引用本文： | 岳华,李明义,马莉,汤承,张兆敏,张斌.基于荧光熔解曲线模式RT-PCR快速检测H5N1亚型禽流感病毒[J].南京农业大学学报,2009,32(4). |
| 作者姓名： | 岳华 李明义 马莉 汤承 张兆敏 张斌 |
| 作者单位： | 1. 西南民族大学生命科学与技术学院,四川 成都 610041;四川省高等院校动物医学重点实验室,四川 成都 610041 2. 中国动物卫生与流行病学中心,山东 青岛,266032 3. 中国医学科学院成都输血研究所,四川 成都,610081 4. 西南民族大学生命科学与技术学院,四川 成都,610041 |
| 基金项目： | 国家"十一五"科技支撑计划项目 |
| 摘 要： | 针对H5N1亚型禽流感病毒(AIV)的HA、NA基因保守序列设计2对引物,建立了SYBR Green Ⅰ双重荧光RT-PCR(RRT-PCR)方法,实现了同一反应管内同时检测AIV的H5和N1亚型基因.熔解曲线分析显示,H5和N1扩增片段的熔解温度分别为(795±03)℃和(833±03)℃,无引物二聚体形成,扩增产物片段大小分别为150bp和474bp,与预期大小相符,测序结果证实为H5和N1亚型基因的靶序列.该方法能从H5N1样本中检出阳性扩增信号,熔解曲线可见H5和N1双特异峰;而H5N2样本有阳性扩增,熔解曲线仅见H5单特异峰.AIV的其他HA亚型和其他病毒的RNA、DNA 无扩增信号.本法最低可检测210拷贝·μL-1和195拷贝·μL-1 H5和N1重组质粒,敏感度分别是RT-PCR的100、1 000倍.本研究建立的基于荧光熔解曲线模式RT-PCR可用于H5N1亚型AIV的检测. |
| 关 键 词： | 禽流感病毒 H5N1亚型 双重荧光RT-PCR 熔解曲线 |
Rapid identification of influenza A virus H5N1 subtype by real-time RT-PCR with melting-curve analysis |
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| YUE Hua,LI Ming-yi,MA Li,TANG Cheng,ZHANG Zhao-min,ZHANG Bin.Rapid identification of influenza A virus H5N1 subtype by real-time RT-PCR with melting-curve analysis[J].Journal of Nanjing Agricultural University,2009,32(4). | |
| Authors: | YUE Hua LI Ming-yi MA Li TANG Cheng ZHANG Zhao-min ZHANG Bin |
| Abstract: | Based on the conserved regions of HA and HA genes of H_5N_1 AIV, two pairs of specific primers were designed and a duplex SYBR Green I -based real-time RT-PCR was developed. This method was able to determine AIV H_5N_1 subtype by detection of H_5 and N_1 gene in a single tube based on melting temperatures ( T_m) discriminations. Two melting p℃aks at temperatures (79. 5±0.3)℃ and (83. 3±0.3)℃ represent H_5 and N_1 gene products, respectively. The PCR products of 150 bp and 474 bp were observed without primer-dimer in agarose gel electrophoresis and confirmed by sequencing. All H_5N_1 strains tested were positive with two specific melting peaks while H_5 N_2 strains showed a single specific peak of H_5 gene. The other subtypes of AIV and viruses yielded no amplification products. The sensitivity of the assay was 21. 0 copies·μL~(-1) for H_5 gene recombinant plasmid and 19. 5 copies ·μL~(-1) for N_1 gene recombinant plasmid, demonstrating 100 and 1 000-fold more sensitive than the conventional RT-PCR method using the same primers. In conclusion, the duplex SYBR Green I -based real-time RT-PCR developed in this study can be used for the detection of H_5N_1 subtype AIV. |
| Keywords: | avian influenza virus H_5N_1 subtype duplex real-time RT-PCR melting curve analysis |
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