中国水产科学2017，Vol.24Issue(6)：1254-1260,7.DOI:10.3724/SP.J.1118.2017.17001
鲁氏耶尔森菌qPCR快速检测方法的建立和应用
Development and application of a rapid qPCR assay for detecting Yersinia ruckeri
摘要
Abstract
Enteric redmouth disease (ERM) is an emerging problem in aquaculture all over the world. ERM is caused by the gram-negative bacterial pathogen Yersinia ruckeri, which can infect salmonids and several other fish taxa and cause clinical signs of hemorrhaging on the body surface and in the intestine. Fish suffering from ERM exhibit exophthalmos, darkening skin, subcutaneous hemorrhage of the mouth and throat, perianal swelling with yellow fluid, and other deleterious outcomes up to and including death. When ERM appears in an aquaculture fa-cility, a large number of fish may be affected over a short period of time. Various antibiotics are available for the treatment of ERM and vaccines can also be used in the treatment and prevention of this disease. Several methods such as ultrastructural examination and LAMP have been developed for ERM detection. However, these assays are generally laborious and time-consuming, and are not sufficiently sensitive. To establish a rapid and quantitative method for the detection of Y. ruckeri, a pair of specific primers was designed and synthesized based on the viru-lent gene rupA. A recombinant plasmid containing part of the rupA gene was used as a standard to construct a standard curve. SYBR Green I real-time quantitative PCR assay was established for the detection of Y. ruckeri by optimizing experimental conditions. The established qPCR method was also applied to the detection of Y. ruckeri in tissues of artificially-infected rainbow trout. Our results showed that the designed primers had good interspeci-fic specificity. The quantitative linear equation was y =-3.3766x+40.012 (R2=0.9958). The detection limit of qPCR method was 57 copies/μL, which is approximately 100-fold greater than conventional PCR. This qPCR method can accurately detect Y. ruckeri in rainbow trout. These results suggest that our qPCR method has the advantages of specificity, sensitivity, rapidity, and quantification, and can be used for rapid diagnosis of early-stage disease and quantitative detection of Y. ruckeri.关键词
鲁氏耶尔森菌/rupA基因/实时荧光定量PCR/肠炎红嘴病/鲑科/虹鳟Key words
Yersinia ruckeri/rupA gene/real-time quantitative PCR/enteric redmouth diseases/Salmonidae/On-corhynchus mykiss分类
农业科技引用本文复制引用
张枭,李绍戊,王荻,曹永生,卢彤岩..鲁氏耶尔森菌qPCR快速检测方法的建立和应用[J].中国水产科学,2017,24(6):1254-1260,7.基金项目
中央级公益性科研院所基本科研业务费专项资助项目(HSY201503) （HSY201503）
新疆维吾尔自治区区域协同创新专项(2016E02052). （2016E02052）
