| 余波，罗永成，徐景峨，周思旋，杨莉，马永兵.大鲵柱状黄杆菌SYBR Green I 荧光定量PCR 诊断试剂盒的研制[J].广东农业科学,2014,41(21):144-148 |
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大鲵柱状黄杆菌SYBR Green I 荧光定量PCR 诊断试剂盒的研制 |
| A SYBR Green I Real-time Quantitative PCR kit for detection of Flavobacterium columnar of giant salamander |
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| DOI： |
| 中文关键词: 大鲵 柱状黄杆菌 荧光定量PCR 16SrRNA基因 |
| 英文关键词: giant salamander Flavobacterium columnar SYBR Green I Real -time Quantitative PCR 16S rRNA gene |
| 基金项目:贵州省科技厅农业攻关项目（黔科合NZ 字[2012]3023 号）；2014 年省级财政渔业发展（大鲵产业）专项资金 |
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| 中文摘要: |
| 根据GenBank中柱状黄杆菌16SrRNA 序列，设计1对特异性引物，PCR 扩增获得16SrRNA 基因片段，并克隆到pMD-18T载体上作为阳性标准品。通过对SYBR Green I荧光定量PCR反应条件的优化，建立了黄杆菌的SYBR Green I荧光定量PCR 诊断方法，以此为基础研制试剂盒。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，对嗜水气单胞菌、荧光假单胞菌、迟缓爱德华、弗氏柠檬酸杆菌均无阳性信号扩增，重复性好，灵敏度可达12 拷贝/μL。结果表明，研制的柱状黄杆菌SYBR Green I实时荧光定量PCR试剂盒具有特异、灵敏、快速、重复性好等特征，适合于大鲵临床样品的检测。 |
| 英文摘要: |
| According to the gene sequences of 16S rRNA gene in GenBank, one pair of specific primer was designed for amplifying the specific fragments of 16S rRNA gene. Then 16S rRNA gene amplified by PCR was cloned into pMD-18T vector and it was used as positive standard. After optimization of annealing temperature and primer concentration, a SYBR Green I Real-time Quantitative PCR was established for detection of Flavobacterium columnar. The melting curve analysis using SYBR Green I dye showed one specific peak, no primer-dimers peak was observed. No amplification was amplified from Aeromonas hydrophila, Edwardsiella tarda, Pseudomonas fluorescens, Citrobacter freundii by the SYBR Green I Realtime Quantitative PCR. The PCR kit was highly sensitive in 12 copies/μL DNA. The results revealed that the SYBR Green I Real-time Quantitative PCR kit was sensitive, specific and it could be used to detect F. columnar in clinical samples. |
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