实时荧光定量PCR扩增特异性vapA基因检测杀鲑气单胞菌摘要【目的.doc

实时荧光定量PCR扩增特异性vapA基因检测杀鲑气单胞菌 摘要: 【目的】为实现杀鲑气单胞菌（Aeromonas salmonicida）早期快速准确定量检测，研究旨在建立杀鲑气单胞菌的 SYBR Green I 实时荧光定量 PCR（real-timePCR）检测方法。【方法】根据GenBank中杀鲑气单胞菌毒力阵列蛋白基因（vapA）保守序列设计并合成一对特异性引物，建立了检测杀鲑气单胞菌的SYBR Green Ⅰ实时荧光定量PCR方法，并对特异性、灵敏度和可重复性进行评价。【结果】结果显示，研究设计的引物具有良好的种间特异性，仅对杀鲑气单胞菌及其亚种有阳性扩增，与其他细菌不发生交叉反应。构建的 real-time PCR 标准曲线质粒拷贝数与循环阈值值呈良好的线性关系，扩增所得标准曲线分别为y = -4.8345x + 42.535，相关系数R2为0.998，最低检测限为34拷贝/μL，较常规PCR的灵敏度高出约1000倍。应用建立的方法检测人工感染的虹鳟病样，15个被检样品呈阳性反应，与细菌常规鉴定方法结果一致。【结论】研究表明，研究建立的基于实时荧光定量PCR技术的杀鲑气单胞菌快速检测方法快速、特异、灵敏，可用于临床诊断和疫病监测。 关键词: 杀鲑气单胞菌；vapA基因；实时荧光定量PCR 中图分类号:S 941. 42 文献标志码:A Real-time PCR Detection of Aeromonas salmonicida by Specific vapA Gene Abstract:【Objective】In order to implement the early and quick quantitative determination of w Aeromonas salmonicida, a SYBR Green I real-time PCR method of Aeromonas salmonicida was established based on the pathogen sequence information.【Method】Based on the vapA of virulence array protein gene sequence of Aeromonas salmonicida, a pair of primers was designed and used in a real time quantitive polymerase chain reaction (qPCR) technique to detect the gene for specific vapA of A.salmonicida. The specificity, sensitivity and repeatability of the system were also evaluated.【Result】 A.salmonicida and its subspecies can be clearly discriminated from the other 10 bacteria species by SYBR Green I real-time qPCR, which indicated that the primer pair has good inter-species specificity. The standard curve established by recombinant plasmid showed a fine linear relationship between initial templates and threshold cycle, which can be described as y = -4.8345x + 42.535 (R2=0.998). The sensitivity analysis showed that the detection limit was 36 copies/μL, which suggested that the sensitivity of real-time qPCR was about 1000 times higher than that of the conventional PCR assay. The established method was applied to detect the samples in rainbow trout after artificial infection. Results showed that 15 of those samples were positive,