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

为实现杀鲑气单胞菌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-time PCR)检测方法.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,对其特异性、灵敏度、可重复性和应用性进行评价.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.研究表明,所建立的基于实时荧光定量PCR技术的杀鲑气单胞菌检测方法快速、特异、灵敏,可用于临床诊断和疫病监测.%In order to implement the early and quick quantitative determination of Aeromonas salmonicida, a SYBR Green I Real-time PCR method of A. salmonicida was established based on the pathogen sequence information. Based on the vapA of virulence array protein gene sequence of A. salmonicida, a pair of primers was designed and used in a real time quantitative polymerase chain reaction (qPCR) assay. The specificity, sensitivity, repeatability and application of the system were also evaluated. The results showed that 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 34 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, which had complete agreement (100%) with bacteriological analysis by isolation and culture. In conclusion, the developed Real-time PCR assay for A. salmonicida is fast, highly specific, and sensitive. This method had a broad application for clinical diagnosis and disease surveillance in aquaculture.