H9亚型鸭源禽流感病毒SYBR GreenⅠ实时荧光定量RT-PCR快速检测方法的建立

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基金项目:

国家科技支撑计划项目(2006BAD06A01);福建省科技计划重点项目(2009N4001);中央高校基本科研业务费专项资金(2010KLEP001);国家973计划项目(2010CB530303)

SYBR GreenⅠ RT-PCR for rapid diagnosis of H9 Subtype Avian Influenza Virus

1.
Institute of Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350013, China

摘要: 针对H9亚型禽流感HA基因保守序列设计引物,建立基于SYBR GreenⅠ检测模式的荧光定量RT-PCR(real-ti me RT-PCR,RRT-PCR),最低检测限为8.33×102拷贝质粒DNA,与常规PCR相比,灵敏度高出100倍。扩增产物的熔解曲线分析只出现1个单特异峰,无引物二聚体,Tm值为(83.83±0.22)℃,组内变异系数为0.52%~1.48%,组间变异系数为0.71%~2.21%,可重复性好;对H5亚型禽流感病毒、鸭肝炎病毒、鸭瘟病毒、鸭源禽1型副粘病毒无扩增;检测速度快,从样本处理到报告结果仅需4.5 h。
- H9亚型禽流感病毒 /
- SYBR GreenⅠ /
- 实时荧光定量PCR(RRT-PCR)
Abstract: A pair of specific primers targeting hemagglutinin gene of H9 avian influenza virus(AIV) was designed for the development of a SYBR Green I fluorescent based real-time RT-PCR(RRT-PCR) to quantify the H9 subtype AIV for rapid diagnosis.The detection limit of RRT-PCR was 8.33×102 plasmid copies.The melting curve analysis using SYBR Green I dye showed one specific peak at the melting temperature of(83.83±0.22)℃ with no primer-dimers peak.No amplification was detected by this method from the unrelated RNA samples,such as H5 subtype AIV,duck hepatitis virus,duck plague virus or avian paramyxovirus type-1.High reproducibility was obtained for detecting plasmid DNA with intra-assay of 0.52%-1.48% and inter-assay of 0.71%-2.21%.
- H9 subtype avian influenza virus /
- SYBR Green I /
- RRT-PCR

[1]	PEIRIS M,YUEN K,LEUNG C,et al.Human infection with influenza H9N2[J].Lancet,1999,354(9182):916-917.
[2]	万春和,傅光华,程龙飞,等.A/Chicken/Zhejiang/HJ/2007(H9N2) 禽流感病毒全基因测序及遗传进化分析[J].农业生物技术学报,2009,17(5):750-757.

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