水产学报2017，Vol.41Issue(12)：1928-1935,8.DOI:10.11964/jfc.20161110608
实时荧光定量PCR扩增特异性vapA基因检测杀鲑气单胞菌
Real-time PCR detection of Aeromonas salmonicida by amplification of specific vapA gene
摘要
Abstract
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.关键词
虹鳟/杀鲑气单胞菌/vapA基因/实时荧光定量PCRKey words
Oncorhynchus mykiss/Aeromonas salmonicida/vapA gene/Real-time qPCR分类
农业科技引用本文复制引用
刘帅,王荻,卢彤岩,曹永生,杨晨,朱国建,李绍戊..实时荧光定量PCR扩增特异性vapA基因检测杀鲑气单胞菌[J].水产学报,2017,41(12):1928-1935,8.基金项目
中央级公益性科研院所基本科研业务费专项(HSY201503) （HSY201503）
新疆维吾尔自治区区域协同创新专项(2016E02052)The Central-level Non-profit Scientific Research Institutes Special Funds (HSY201503) （2016E02052）
Xinjiang Uygur Autonomous Region Collaborative Innovation Project (2016E02052) （2016E02052）
