| 大鲵致病性嗜水气单胞菌SYBR GreenⅠ荧光定量PCR诊断试剂盒的研制 |
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| 引用本文： | 余波,;罗永成,;马永兵,;徐景峨,;周思旋,;杨莉,;史开志.大鲵致病性嗜水气单胞菌SYBR GreenⅠ荧光定量PCR诊断试剂盒的研制[J].广西农业生物科学,2014(5):1098-1103. |
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| 作者姓名： | 余波 ;罗永成 ;马永兵 ;徐景峨 ;周思旋 ;杨莉 ;史开志 |
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| 作者单位： | [1]贵州省畜牧兽医研究所,贵阳550005; [2]贵州省水产技术推广站,贵阳550001; [3]贵阳市水产技术推广站,贵阳550004 |
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| 基金项目： | 贵州省科技厅农业攻关项目（黔科合NZ字[2012]3023号）资助 |
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| 摘 要： | 根据GenBank中致病性嗜水气单胞菌特异性的气溶素基因序列,设计1对引物,利用普通PCR技术扩增获得hlyA基因片段,并克隆到pMD-18T载体上作为阳性标准品。通过对SYBR GreenⅠ荧光定量PCR反应条件的优化,建立了快速检测致病性嗜水气单胞菌的SYBR GreenⅠ荧光定量诊断方法,以此为基础研制出试剂盒。试剂盒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,对非致病性嗜水气单胞菌、弗氏柠檬酸杆菌、迟缓爱德华菌、柱状黄杆菌均无阳性信号扩增,重复性好,灵敏度可达1.0x101拷贝/uL。结果表明研制的致病性嗜水气单胞菌SYBR GreenⅠ实时荧光定量PCR试剂盒具有特异、灵敏、快速、重复性好等特点,适合于大鲵临床样品的检测。
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| 关 键 词： | 致病性嗜水气单胞菌 荧光定量PCR 气溶素基因 |
A SYBR Green I Real-time Quantitative PCR Kit for Detection of Pathogenic A eromonas hydrophila |
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| Institution: | Yu Bo Luo Yongcheng Ma Yongbing Xu Jing＇e Zhou Sixuan Yang Li Shi Kaizhi(1. Guizhou Instituteof Animal Husbandry and Veterinary Medicine, Guiyang, 550005; 2 Fishery Technical Extension Station of Guizhou Province, Guiyang, 550001; 3 Fishery Technical Extension Station of Guiyang Province, Guiyang, 550004) |
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| Abstract: | According to the gene sequences ofhlyA gene in GenBank, one pairs of specific primer was designed for amplifying the specific fragments ofhlyA gene. The amplified hlyA gene fragment was cloned into pMD- 18T vector and used as positive standard. After optimization of annealing temperature and primers concentrations, a SYBR Green I Real-time Quantitative PCR was established for simultaneous detection of Aeromonas hydrophila. The melting curve analysis showed one specific peak, a no primer-dimers peak, was observed. No amplification was amplified from nonpathogenetic A. hydrophila, Citrobacter freundii, Edwardsiella tarda, Flavobacterium by the SYBR Green I Real-time Quantitative PCR. The PCR kit was highly sensitive in 1.0×10-1 copies/μL DNA. The results revealed that the SYBR Green I Real-time Quantitative PCR kit was specific, sensitive, fast and good re- peatability. It could be used to detect Aeromonas hrdrot）hila in clinical samoles. |
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| Keywords: | Pathogenic A eromonas hydrophila SYBR Green I Real -time Quantitative PCR hlyA gene |
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